Effect of angiotensin II on artificial lipid membranes

(5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between $\text{Δ>G = 5 · 10}{}^{\mathrm{?11}}\text{Ω}{}^{\mathrm{?}}$ and 3.5 · 10^?10 Ω^?1. These fluctuations depend on the voltage and the hydrostatic pressure. The membrane resistance is lowered by $\text{Δ>R = 6.1 · 10}{}^7\text{Ω · cm}{}^2$. With (5-isoleucine, 8-leucine)-angiotensin II the jumps are of a single amplitude ($\text{Δ>G = 2 · 10}{}^{\mathrm{?10}}\text{Ω}{}^{\mathrm{?1}}$). In both cases water and ions are transported across the membrane.     

Age-dependent activation of PKC isoforms by angiotensin II in the proximal nephron

ANG II increases fluid absorption in proximal tubules from young rats more than those from adult rats. ANG II increases fluid absorption in the proximal nephron, in part, via activation of protein kinase C (PKC). However, it is unclear how age-related changes in ANG II-induced stimulation of the PKC cascade differ as an animal matures. We hypothesized that the response of the proximal nephron to ANG II decreases as rats mature due to a reduction in the amount and activation of PKC rather than a decrease in the number or affinity of ANG II receptors. Because PKC translocates from the cytosol to the membrane when activated, we first measured PKC activity in the soluble and particulate fractions of proximal tubule homogenates exposed to vehicle or 10(-10) M ANG II from young (26 +/- 1 days old) and adult rats (54 +/- 1 days old). ANG II increased PKC activity to the same extent in homogenates from young rats (from 0.119 +/- 0.017 to 0.146 +/- 0.015 U/mg protein) (P < 0.01) and adult rats (from 0.123 +/- 0.020 to 0.156 +/- 0.023 U/mg protein) (P < 0.01). Total PKC activity did not differ between groups (0.166 +/- 0.018 vs. 0.181 +/- 0.023). We next investigated whether activation of the alpha-, beta-, and gamma-PKC isoforms differed by Western blot. In homogenates from young rats, ANG II significantly increased activated PKC-alpha from 40.2 +/- 6.5 to 60.2 +/- 9.5 arbitrary units (AU) (P < 0.01) but had no effect in adult rats (46.1 +/- 5.1 vs. 48.5 +/- 8.2 AU). Similarly, ANG II increased activated PKC-gamma in proximal tubules from young rats from 47.9 +/- 13.2 to 65.6 +/- 16.7 AU (P < 0.01) but caused no change in adult rats. Activated PKC-beta, however, increased significantly in homogenates from both age groups. Specifically, activated PKC-beta increased from 8.6 +/- 1.4 to 12.2 +/- 2.1 AU (P < 0.01) in homogenates from nine young rats and from 19.0 +/- 5.5 to 25.1 +/- 7.1 AU (P < 0.01) in homogenates from 12 adult rats. ANG II did not alter the amount of soluble PKC-alpha, -beta, and -gamma significantly. The total amount of PKC-alpha and -gamma did not differ between homogenates from young and adult rats, whereas the total amount of PKC-beta was 59.7 +/- 10.7 and 144.9 +/- 41.8 AU taken from young and adult rats, respectively (P < 0.05). Maximum specific binding and affinity of ANG II receptors were not significantly different between young and adult rats. We concluded that the primary PKC isoform activated by ANG II changes during maturation.     

ATP directly enhances calcium channels in the luminal membrane of the distal nephron.

Calcium (Ca(2+)) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca(2+) channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca(2+) uptake by these vesicles loaded with ATP or the carrier. The presence of 100 microM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca(2+) uptake from 0.57 +/- 0.02 to 0.71 +/- 0.02 pmol/microg per 10 sec (P < 0. 01) in the absence of Na(+) and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/microg per 10 sec (P < 0.01) in the presence of 100 mM Na(+). This effect was dose dependent with an EC(50) value of approximately 40 microM. ATP action involved the high-affinity component of Ca(2+) transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPgammas abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca(2+) transport. Inclusion of 20 microM cytochalasin B decreased 0.5 mM Ca(2+) uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/microg per 10 sec (P< 0.01). However, when both 100 microM ATP and 20 microM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca(2+) uptake by the PT luminal membrane. We conclude that the high-affinity Ca(2+) channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca(2+) channels within this membrane.     

Characterization of three types of calcium channel in the luminal membrane of the distal nephron

We previously reported a dual kinetics of Ca2+ transport by the distal tubule luminal membrane of the kidney, suggesting the presence of several types of channels. To better characterize these channels, we examined the effects of specific inhibitors (i.e., diltiazem, an L-type channel; omega-conotoxin MVIIC, a P/Q-type channel; and mibefradil, a T-type channel antagonist) on 0.1 and 0.5 mM Ca2+ uptake by rabbit nephron luminal membranes. None of these inhibitors influenced Ca2+ uptake by the proximal tubule membranes. In contrast, in the absence of sodium (Na+), the three channel antagonists decreased Ca2+ transport by the distal membranes, and their action depended on the substrate concentrations: 50 microM diltiazem decreased 0.1 mM Ca2+ uptake from 0.65 +/- 0.07 to 0.48 +/- 0.06 pmol. microg-1.10 s-1 (P < 0.05) without influencing 0.5 mM Ca2+ transport, whereas 100 nM omega-conotoxin MVIIC decreased 0.5 mM Ca2+ uptake from 1.02 +/- 0.05 to 0.90 +/- 0.05 pmol. microg-1.10 s-1 (P < 0.02) and 1 microM mibefradil decreased it from 1.13 +/- 0.09 to 0.94 +/- 0.09 pmol. microg-1.10 s-1 (P < 0.05); the latter two inhibitors left 0.1 mM Ca2+ transport unchanged. Diltiazem decreased the Vmax of the high-affinity channels, whereas omega-conotoxin MVIIC and mibefradil influenced exclusively the Vmax of the low-affinity channels. These results not only confirm that the distal luminal membrane is the site of Ca2+ channels, but they suggest that these channels belong to the L, P/Q, and T types.     

Fluoride reabsorption by nonionic diffusion in the distal nephron of the dog

This study was done to test the hypothesis that fluoride reabsorption is extensive from the distal nephron, the major site for tubular fluid acidification, and to compare the distal nephron handling of fluoride and chloride. Ten stop-flow studies were done in five dogs anesthetized with pentobarbital. Urinary alkalinization was achieved by the intravenous infusion of sodium bicarbonate and acetazolamide or lithium chloride. Acidification was achieved by the infusion of sodium nitrate or sodium sulfate. The results indicate that the extent of fluoride reabsorption from the distal nephron is inversely correlated with urinary pH (P less than 0.001). When the urine was strongly acidified by the infusion of sodium sulfate, urine to plasma fluoride concentration ratios were less than 1.0, a finding not previously reported from studies of the renal handling of fluoride. The reabsorption of fluoride from the distal nephron was not correlated consistently with that of chloride. The results indicate that the distal nephron is an important site for the reabsorption of fluoride and they provide additional evidence that HF is the permeating moiety.     

Influence of intrarenally generated angiotensin II on renal hemodynamics and tubular reabsorption.

There is growing recognition that angiotensin II (ANG II) formed intrarenally exerts direct effects on renal hemodynamics and tubular reabsorption. In vivo micropuncture experiments performed in anesthetized rats have shown that peritubular capillary infusion of either ANG II or angiotensin I (ANG I), at rates that do not markedly influence baseline vascular resistance, can increase proximal tubular reabsorption rate and enhance the responsiveness of the tubuloglomerular feedback mechanism. With higher ANG II or ANG I infusion rates, pronounced preglomerular vasoconstriction occurs, resulting in reduced glomerular capillary pressure and single nephron glomerular filtration rate. The effects of peritubular capillary infusion of ANG I on glomerular function have been shown to be inhibited by the ANG II receptor antagonist, saralasin, indicating that the observed effects of ANG I on proximal tubular reabsorption and glomerular function are not due to direct effects of the decapeptide but are mediated by increases in the interstitial ANG II concentrations resulting from intrarenally generated ANG II. Interestingly, neither peritubular capillary infusion nor systemic administration of large doses of the angiotensin-converting enzyme (ACE) inhibitor, enalaprilat, elicited significant blockade of the single nephron hemodynamic responses to peritubular infusion of ANG I. These findings indicate that intrarenal conversion of ANG I to ANG II occurs, at least in part, at a site which is inaccessible to acutely administered ACE inhibitors, or that there is an alternative pathway for the intrarenal conversion of ANG I to ANG II that is not blocked by ACE inhibitors.     

Effects of angiotensin on proximal tubular reabsorption

Effects of angiotensin II on rat, rabbit, and bovine proximal tubular reabsorption have been demonstrated with a variety of techniques, including in vivo microperfusion, free-flow micropuncture of surface and juxtamedullary nephrons, perfusion of isolated tubules in vitro, and cell culture. Blockade of endogenous angiotensin production in vivo with converting-enzyme inhibition, or of receptors with saralasin, consistently inhibits proximal reabsorption of fluid in both superficial and juxtamedullary proximal tubules. Angiotensin effects on the proximal tubule are not neurally mediated, for they persist in denervated kidneys and are seen in nerve-free isolated tubules. Physiological concentrations of angiotensin (10(-11)-10(-9) M) stimulate electroneutral sodium transport from the basolateral membrane, whereas pharmacological doses (10(-7) M and above) inhibit reabsorption. The stimulatory effects appear to be receptor mediated. In addition to these direct effects of angiotensin on the proximal tubule epithelium, endogenous angiotensin may also alter peritubular physical forces to further enhance proximal reabsorption. These effects of angiotensin may represent an important homeostatic mechanism during states of extracellular fluid volume depletion.     

Effect of angiotensin II non-peptide AT(1) antagonist losartan on phosphatidylethanolamine membranes

Losartan was found to affect both the thermotropic behavior and molecular mobility of dimyristoyl- and dipalmitoyl-phosphatidylcholine membranes (Theodoropoulou and Marsh, Biochim. Biophys. Acta 1461 (1999) 135-146). At low concentrations, the antagonist is located close to the interfacial region of the phosphatidylcholine bilayer while at high mole fractions it inserts deeper in the bilayers. In the present study, we investigated the interactions of losartan with phosphatidylethanolamine membranes using differential scanning calorimetry (DSC), electron spin resonance (ESR) and 31P nuclear magnetic resonance (NMR) spectroscopy. DSC showed that the antagonist affected the thermotropic transitions of dimyristoyl-, dipalmitoyl- and dielaidoyl-phosphatidylethanolamine membranes (DMPE, DPPE and DEPE, respectively). ESR spectroscopy showed that the interaction of losartan with phosphatidylethanolamine membranes is more superficial than in the case of phosphatidylcholine bilayers. Additionally, losartan increased the spin-spin broadening of 12-PESL spin labels in the gel phase of DMPE and DPPE membranes, while in the case of DEPE membranes the opposite effect was observed. (31)P-NMR showed that the antagonist stabilizes the fluid lamellar phase of DEPE membranes relative to the hexagonal H(II) phase. Our results show that losartan affects the thermotropic behavior of phosphatidylethanolamine membranes, while the molecular mobility of the membranes is not affected greatly. Furthermore, its interactions with phosphatidylethanolamine membranes are more superficial than with phosphatidylcholine bilayers.     

血管紧张素Ⅱ对模拟缺血心室肌细胞L-型钙通道的影响

实验研究了血管紧张素II（AngⅡ)对模拟缺血心室肌细胞L－型钙离子通道的作用,用胶原酶酶解法急性分离豚鼠心室肌细胞,以全细胞膜片钳方法记录心室肌细胞的L－型钙电流（ICa L.)。采用低氧,无糖,高乳酸和酸中毒综合方式模拟缺血液灌流,造成心室肌细胞的模拟缺血,并在缺血的基础上继续用含100mmol/A AngⅡ灌流细胞,观察AngⅡ对模拟缺血心室肌细胞钙离子通道的影响,实验结果显示,模拟缺血时ICa.L峰值电流明显减小,最大激活电压为0mV,AngⅡ能抵抗模拟缺血对ICa,L的抑制效应,使ICa,L峰值电流增大,并使最大激活电压左移至－10mV。     

Effect of angiotensin II on RNA synthesis by isolated nuclei

Peptide hormones are known to bind to cell surface receptors as the first step in the generation of their effects on target tissues. However, it remains uncertain whether internalized hormone might also play a role in the production of longterm or trophic effects of peptide hormones. Because the peptide hormone angiotensin II appears to be internalized by target cells, we studied the effect of this peptide on isolated hepatic nuclei. At both 5 X 10(-7)M and 5 X 10(-9)M, angiotensin II significantly increased RNA synthesis. This effect was not mimicked by Sar1-Ala8-angiotensin II (saralasin) or the unrelated nonapeptide teprotide.     

Secretion and reabsorption of uterine luminal fluid in rats

Treatment of ovariectomized rats with oestradiol-17beta and progesterone demonstrated that oestradiol-17beta causes secretion of sodium, potassium and water into the lumen of the uterine horn and that progesterone causes reabsorption of these substances.     

Tyrosine kinase activation by the angiotensin II receptor in the absence of calcium signaling

The angiotensin II type 1 (AT(1)) receptor signals via heterotrimeric G-proteins and intracellular tyrosine kinases. Here, we investigate a modified AT(1) receptor, termed M5, where the last five tyrosines (residues 292, 302, 312, 319, and 339) within the intracellular carboxyl tail have been mutated to phenylalanine. This receptor did not elevate cytosolic free calcium or inositol phosphate production in response to angiotensin II, suggesting an uncoupling of the receptor from G-protein activation. Despite this, the M5 receptor still activated tyrosine kinases, induced STAT1 tyrosine phosphorylation, and stimulated cell proliferation. We also studied another AT(1) mutant receptor, D74E, stably expressed in Chinese hamster ovarian cells and a fibroblast cell line from mice with a genetic inactivation of Galpha(q/11). Both cell lines have a deficit in calcium signaling and in G-protein activation, and yet in both cell lines, angiotensin II induced the time-dependent tyrosine phosphorylation of STAT1. These studies are the first to show the ability of a seven-transmembrane receptor to activate intracellular tyrosine kinase pathways in the absence of a G-protein-coupled rise in intracellular calcium.     

Reversal of hyperglycemic preconditioning by angiotensin II: role of calcium transport

Myocardial cell death is an important contributor to the development of diabetic cardiomyopathy. It has been proposed that diabetes-mediated upregulation of the renin-angiotensin system leads to oxidative stress, the trigger for cardiomyocyte death and contractile dysfunction. However, the adverse effect of ANG II on the diabetic heart may extend beyond the development of the cardiomyopathy. ANG II also alters specific modulators of ischemic injury, such as PKC and calcium transport. Therefore, the present study examined the effect of ANG II on hyperglycemic preconditioning, a glucose-mediated condition associated with the elevation of PKC activity and alterations in calcium transport that render the cell resistant to hypoxia. Exposure of the glucose-treated cell to ANG II during the prehypoxic period blocked glucose-mediated cardioprotection. The reversal of hyperglycemic preconditioning was associated with enhanced accumulation of Ca(2+) during hypoxia, an effect prevented by inhibition of the Na(+)/ H(+) exchanger and the T-type Ca(2+) channel. The inhibitors of hypoxia-mediated Ca(2+) accumulation also blocked the reversal of hyperglycemic preconditioning by ANG II. Thus ANG II and glucose treatment exert opposite actions on the Na(+)/ H(+) exchanger and the T-type Ca(2+) channel. Because those transporters are involved in hypoxia-mediated apoptosis, they are logical candidates for the beneficial effects of high glucose and the adverse effects of ANG II on the hypoxic cardiomyocyte.     

Effect of angiotensin II on liposome uptake by the rat brain in vivo

Studies have been performed to assess the possibility of using small unilamellar liposomes as therapeutic carriers to the brain of hypertensive rats. Rats were made temporal hypertensive by the infusion of angiotensin II (AII; 15 micrograms in 1 ml) through their right common carotid artery. Another control group was infused with physiological saline. Free 125iodine-BSA (125I-BSA) and 125I-BSA encapsulated liposomes (average diameter approximately equal to 100 nm) were injected in the tail vein 2 min after the infusion of AII or saline. Plasma radioactivity was monitored at different times up to 15 min when the cerebral uptake of 125I-BSA was determined. While a little variation in plasma clearance pattern of liposomes in hypertensive and control group was noticed, the uptake by cerebral tissues was markedly higher in hypertensive group. Analysis of pharmacokinetic parameters in relation to cerebral uptake indicated AII induced a short term opening of the blood-brain barrier (BBB) resulting in an increased cerebral uptake. Positively charged liposomes was found to be most effective in hypertensive state.     

吗啡和血管紧张素II对大鼠脑突触小体Ca^2＋摄取的拮抗效应

Behavioral observations have repeatedly shown that the analgesic effect of morphine can be antagonized by intracerebroventricular injection of angiotensin I (A I), although mechanisms underlying the action were obscure. Since a prevention of Ca2+ uptake into the nerve terminals was considered as one of the mechanisms for morphine analgesia, we examined the effect of A I and morphine on the 45Ca uptake by rat brain synaptosomal preparations. Morphine of 10(-8)-10(-6) mol/L produced a dose-related suppression on synaptosomal 45Ca uptake, which was completely reversed by the opioid antagonist naloxone of 10(-6) mol/L. A I of 10(-8)-10(-6) mol/L, on the contrary, enhanced 45Ca uptake. This effect was totally abolished by saralasin, a A I antagonist, at 10(-6) mol/L. When synaptosomal preparations were incubated in a mixture of morphine (10(-6) mol/L) and A I (10(-8)-10(-6) mol/L), the effect of morphine was almost completely reversed. The results suggest that the distinct effect of A I may account for, at least in part, the antagonistic effect of A I on morphine analgesia.     

Stimulation of glycogenolysis in hepatocytes by angiotensin II may involve both calcium release and calcium influx

The stimulation of [³H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca-mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca-influx is also involved in the mechanism of Ca-mobilization by these agents in the guinea pig hepatocyte.     

Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration

Secretin is a vasoactive peptide capable of acting on transmembrane volume fluxes. We measured nephron filtration (SNGFR) and resorption during secretin microinjection (MIJ) into the tubular lumen or microperfusion (MP) into peritubular capillaries. In 24 rat nephrons, SNGFR, measured by collections from the distal tubule, rose from 25+/- 4 to 61+/-8 nl/min during MIJ of saline containing secretin 10(-9) M into the last convolution of the proximal tubule (LP). Percent and absolute resorptions rose from 70 to 90% and from 20+/-4 to 56+/-8 nl/min, respectively. During MIJ of secretin, 3 x 10(-)(9) M into the first convolution of the proximal tubule, SNGFR, measured at LP, rose from 32+/-4 to 61+/-8 nl/min, percent and absolute reabsorptions from 52+/-4 to 78+/-3% and from 16+/-2 to 50+/-7 nl/min, respectively (n = 30). During MP of secretin, 1.5x10(-9) M, SNGFR fell from 39+/-6 to 15+/-4, resorption from 19+/-4 to 9+/-2 nl/min, while percent resorption rose from 43+/-6 to 59+/-5% (n = 15). While all MIJ and MP changes were significant (P<0.001), paired pre- versus post-MIJ and MP values were not. Secretin is a powerful vasoconstrictor when perfused into peritubular capillary blood, unlike systemic and intra-arterial injections. When injected into the tubular lumen, it up-regulates SNGFR and increases reabsorption directly.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.