Distribution of Non-AT1, Non-AT2 Binding of 125I-Sarcosine1, Isoleucine8 Angiotensin II in Neurolysin Knockout Mouse Brains

The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of ¹²⁵I-Sarcosine¹, Isoleucine⁸ Ang II (¹²⁵I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) ¹²⁵I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. ¹²⁵I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT₁, non-AT₂, non-neurolysin Ang II binding site in the mouse brain. Binding of ¹²⁵I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT₁, non-AT₂, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT₂ receptor binding in the neurolysin knockout brain and a trend towards decreased AT₁ receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (−2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.     

Modulation of delayed rectifier potassium current by angiotensin II in CATH.a cells

Angiotensin II (Ang II) modulates, via Ang II type 1 (AT(1)) receptors, the activity of brain catecholaminergic neurons. Here we utilized catecholaminergic CATH.a cells to define the effects of Ang II on delayed rectifier K(+) current (I(Kv)), one of the factors that determines changes in neuronal activation. Receptor binding analyses demonstrated the presence of AT(1) receptors in CATH.a cells. Whole cell voltage clamp experiments in these cells revealed that Ang II (100nM) produced a significant inhibition of I(Kv), that was abolished by the AT(1) receptor blocker, losartan (1 microM), or by inhibition of phospholipase C (PLC) with U73122 (10 microM). Furthermore, this action of Ang II was completely abolished by co-inhibition of protein kinase C (PKC) and calcium/calmodulin protein kinase II (CaMKII). These results demonstrate that Ang II produces an inhibition of I(Kv) in CATH.a cells, via an intracellular pathway that includes PLC, PKC, and CaMKII.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

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.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

PDGF-β receptor and PKC have no effect on angiotensin II-induced JAK2 and STAT1 phosphorylation in vascular smooth muscle cells under high glucose condition

Background: The mechanisms responsible for the accelerated cardiovascular disease in diabetes, as well as the increased hypertrophic effects of angiotensin II (Ang II) under hyperglycemic condition, are not very clear. Evidences show that platelet-derived growth factor (PDGF) and protein kinase C (PKC) play a critical role in this effect. In our study, we examined the role of PKC and PDGF receptor on JAK2 and STAT1 phosphorylation under high glucose (HG) condition (25 mmol/L) in response to Ang II in cultured vascular smooth muscle cells (VSMC).

Methods: VSMCs were isolated from the thoracic aorta of male Wistar rats and were cultured. Growth-arrested VSMCs were placed in either normal glucose (NG) or HG condition for 48?h and then VSMCs were stimulated with agonists and antagonists. The tyrosine phosphorylation of JAK2 or STAT were determined by immunoblotting using specific antibodies.

Results: High glucose markedly increased the phosphorylation of tyrosine residues of JAK2 and serine residues of STAT 1 compared with cells cultured in NG (5.5 mmol/L) with and without Ang II stimulation. Experiments made with specific PDGF-β receptor inhibitor AG1295 and PKC inhibitor GF109203X showed that there were no changes in Ang II-stimulated JAK2 and STAT1 phosphorylation under NG and HG conditions compared with experiments without inhibitors.

Conclusion: According to our findings, Ang II-stimulated JAK2 and STAT1 phosphorylation under either NG or HG condition do not proceed via a different pathway rather than PKC and PDGF-β 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.     

Angiotensin type 1 receptor is linked to inhibition of nitric oxide production in pulmonary endothelial cells

We previously demonstrated that angiotensin II (Ang II) stimulates an increase in nitric oxide synthase (NOS) mRNA levels, eNOS protein expression and NO production via the type 2 (AT2) receptor, whereas signaling via the type 1 (AT1) receptor negatively regulates NO production in bovine pulmonary artery endothelial cells (BPAECs). In the present study, we investigated the components of the AT1 receptor-linked signaling pathway(s) that are involved in the downregulation of eNOS protein expression in BPAECs. Treatment of BPAECs with either AT1 receptor antagonists or an anti-AT1 receptor antibody induced eNOS protein expression. Furthermore, intracellular delivery of GP-Antagonist-2A, an inhibitor of Galphaq proteins, and treatment of BPAECs with U73122, a phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, enhanced eNOS protein expression. Treatment of BPAECs with the cell-permeable calcium chelator, BAPTA/AM, increased eNOS protein expression at 8 h, while increasing intracellular calcium with either thapsigargin or A23187 prevented Ang II-induced eNOS protein expression. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, completely prevented Ang II-stimulated eNOS protein expression at 8 h, whereas depletion of PKC by long-term treatment with PMA, induced eNOS protein expression. Treatment of BPAECs with a PKCalpha-specific inhibitor or transfection of BPAECs with an anti-PKCalpha neutralizing antibody stimulated eNOS protein expression. Conversely, rottlerin, a PKCdelta specific isoform inhibitor had no effect on basal or Ang II-stimulated eNOS protein expression. Moreover, treatment of BPAECs with U73122, BAPTA/AM and PKCalpha-specific inhibitors increased NO production at 8 h. In conclusion, Ang II downregulates eNOS protein expression via an AT1 receptor-linked pathway involving Galphaq/PLC/calcium/PKCalpha signaling pathway in BPAECs.     

Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.     

Renal nuclear angiotensin II receptors in normal and hypertensive rats

Accumulation of Angiotensin II (Ang II) in the kidneys of hypertensive rats infused chronically with Ang II occurs by AT1 receptor mediated internalization of Ang II, which may interact with intracellular targets, including nuclear binding sites. The aims of this study were to determine if kidney cell nuclei have specific Ang II binding sites and if chronic infusion of Ang II (70 ng/min; n=9) influences the nuclear Ang II binding capacity. Kidneys were harvested from control and Ang II infused rats and the renal cortexes were homogenized to obtain crude membrane preparations and nuclear fractions. Ang II binding sites were measured with a single point assay by incubating each fraction with 10 nM 125I-Sar-Ile-Ang II in the absence (total binding sites) or presence of either 2.5 M Sar-Leu-Ang II or 25 microM losartan to detect specific AT or AT1 binding sites. Both fractions exhibited specific Ang II binding sites that were displaced by both saralasin and losartan. In control rats, crude membrane preparations had 792 +/- 218 and the nuclear fraction had 543 +/- 222 fmol/mg protein AT1 receptors. AT1 receptor levels in membrane (885 +/- 170 fmol/mg protein) and nuclear fractions (610 +/- 198 fmol/mg protein) were not significantly different in Ang II infused rats. These data support the presence of nuclear Ang II receptors predominantly of the AT1 subtype in renal cells. Chronic Ang II infusion did not alter overall Ang II receptor densities.     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.     

Protein Kinase C Agonists Increase the Expression of Angiotensin II Receptors in Neuronal Cultures

Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by alpha 1-adrenergic receptors. Because alpha 1-adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1-day-old rat brains. The active phorbol ester phorbol-12-myristate-13-acetate (TPA) caused time- and concentration-dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester-induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.     

Lack of alpha-1-adrenergic receptor-mediated downregulation of angiotensin II receptors in neuronal cultures from spontaneously hypertensive rat brain

Neuronal cells from Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rat brains were established in culture to compare the expression of angiotensin II (Ang II) specific receptors and their regulation by norepinephrine (NE). Neurons from SH rat brains possess twice more Ang II specific receptors and expressed a proportional increase in Ang II stimulated [³H]-NE uptake compared with WKY neurons. NE caused a dose-dependent decrease in¹²⁵I-Ang II binding in WKY neurons, an effect not observed when neurons from SH rat brains were incubated with NE. These observations suggest that the lack of NE-induced downregulation of Ang II receptors in neuronal cultures is genetically regulated.     

Angiotensin II Activates Programmed Myocyte Cell Deathin Vitro

To determine whether angiotensin II (Ang II) can induce apoptosis of neonatal ventricular myocytes, these cells were exposed to 10⁻⁹MAng II for 24 hin vitroand the effects of this intervention on programmed myocyte cell death were examined by the terminal deoxynucleotidyl transferase assay and DNA gel electrophoresis. Ang II resulted morphologically in a 2.5-fold increase in the percentage of myocytes with double strand cleavage of the DNA and biochemically in the formation of DNA fragments equal in size to mono- and oligonucleosomes. Moreover, Ang II stimulation was characterized by a 37% increase in resting level of intracellular calcium and the activation of calcium-dependent endogenous endonuclease. In contrast, pH-dependent endogenous endonuclease was not enhanced by the addition of Ang II. Ang II-induced DNA damage was inhibited by the AT₁receptor antagonist, losartan. Similarly, the calcium chelator, BAPTA-AM, prevented Ang II-mediated cell death. Conversely, the calcium ionophore, A23187, triggered programmed cell death. Finally, the selective AT₂receptor subtype blocker, PD123319, failed to reduce myocyte apoptosis. In conclusion, ligand binding of AT₁receptors may initiate programmed myocyte cell death via an elevation in cytosolic calcium and the stimulation of calcium-dependent endogenous endonuclease.     

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT₁. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10^–11–10^–9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT_1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [¹²⁵I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT_1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [¹²⁵I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein.An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT_1A receptor transfected CHO-K1 cells, angiotensin II (10^–9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT_1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT_1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.     

Autoradiographic localization and characterization of angiotensin II binding sites in the spleen of rats and mice

Specific binding sites for angiotensin II (Ang II) were localized in the red pulp of the spleen of rats and mice by quantitative autoradiography using 125I-Sar1-Ang II as a ligand. In the rat, the binding was saturable and specific, and the rank order for Ang II derivatives as competitors of 125I-Sar1-Ang II binding correlates well with their affinity for Ang II receptors in other tissues. Kinetic analysis in the rat spleen revealed a single class of binding sites with a KD of 1.11 nM and a Bmax value of 81.6 fmol/mg protein. Ang II binding sites were also localized on isolated rat spleen cells with similar affinity but with much lower Bmax, 9.75 fmol/mg protein. Ang II receptors were not detected in thymus sections from rats or mice, or on isolated rat thymocytes. The binding sites described here might represent a functional Ang II receptor with a role in the regulation of splenic volume and blood flow and in the modulation of the lymphocyte function.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.