The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT₁ and AT₄ receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of ¹²⁵I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ～1?nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT₁ and AT₄ receptor antagonists, respectively). The insulin release was accompanied by a ⁴⁵Ca²⁺ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT₄ receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT₁ and AT₄ receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca²⁺. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.     

Fibronectin synthesis by high glucose level mediated proliferation of mouse embryonic stem cells: Involvement of ANG II and TGF‐β1

The role of individual supplements necessary for the long‐term self‐renewal of embryonic stem (ES) cells is poorly characterized in feeder/serum‐free culture systems. This study sought to characterize the relationship between the effects of glucose on ES cell proliferation and fibronectin (FN) synthesis, and to assess the mechanisms responsible for these cellular effects of glucose. Treatment of the two ES cells (ES‐E14TG2a and ES‐R1) with 25 mM glucose (high glucose) increased the expression levels of FN mRNA and protein. In addition, high glucose and ANG II synergistically increased FN expression level, which coincident with data showing that high glucose increased the mRNA expression of angiotensin II (ANG II) type 1 receptor (AT₁R), angiotensinogen, and FN, but not ANG II type 2 receptor. High glucose also increased the intracellular calcium (Ca²⁺) concentration and pan‐protein kinase C (PKC) phosphorylation. Inhibition of the Ca²⁺/PKC pathway blocked high glucose‐induced FN expression. High glucose or ANG II also synergistically increased transforming growth factor‐beta1 (TGF‐β₁) expression, while pretreatment with losartan abolished the high glucose‐induced increase in TGF‐β₁ production. Moreover, TGF‐β₁‐specific small interfering RNA inhibited high glucose‐induced FN expression and c‐Jun N‐terminal kinase (JNK) activation. The JNK inhibitor SP600125 blocked high glucose‐induced FN expression and inhibited cell cycle regulatory protein expression induced by high glucose or TGF‐β₁. In this study, inhibition of AT₁R, Ca²⁺/PKC, TGF‐β₁, JNK, FN receptor blocked the high glucose‐induced DNA synthesis, increased the cell population in S phase, and the number of cells. It is concluded that high glucose increases FN synthesis through the ANG II or TGF‐β1 pathways, which in part mediates proliferation of mouse ES cells. J. Cell. Physiol. 223: 397–407, 2010. © 2010 Wiley‐Liss, Inc.     

Modulation of KCa3.1 Channels by Eicosanoids,Omega-3 Fatty Acids,and Molecular Determinants

Background

Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modula-tion of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca²⁺/calmodulin-regulated K⁺ channels (K_Ca3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade.

Methodology/Principal Findings

We studied modulation of cloned human hK_Ca3.1 and the mutant hK_Ca3.1^V275A in HEK-293 cells, of rK_Ca3.1 in aortic endothelial cells, and of mK_Ca3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca²⁺-activated hK_Ca3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K_Ca3.1^V275A mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK_Ca3.1 and rK_Ca3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K_Ca3.1 currents.

Conclusions/Significance

Inhibition of K_Ca3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids.     

Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II

The present study examined the effects of ANG II on the renal synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and its contribution to the renal vasoconstrictor and the acute and chronic pressor effects of ANG II in rats. ANG II (10(-11) to 10(-7) mol/l) reduced the diameter of renal interlobular arteries treated with inhibitors of nitric oxide synthase and cyclooxygenase, lipoxygenase, and epoxygenase by 81 +/- 8%. Subsequent blockade of the synthesis of 20-HETE with 17-octadecynoic acid (1 micromol/l) increased the ED(50) for ANG II-induced constriction by a factor of 15 and diminished the maximal response by 61%. Graded intravenous infusion of ANG II (5-200 ng/min) dose dependently increased mean arterial pressure (MAP) in thiobutylbarbitol-anesthetized rats by 35 mmHg. Acute blockade of the formation of 20-HETE with dibromododecenyl methylsulfimide (DDMS; 10 mg/kg) attenuated the pressor response to ANG II by 40%. An intravenous infusion of ANG II (50 ng. kg(-1). min(-1)) in rats for 5 days increased the formation of 20-HETE and epoxyeicosatrienoic acids (EETs) in renal cortical microsomes by 60 and 400%, respectively, and increased MAP by 78 mmHg. Chronic blockade of the synthesis of 20-HETE with intravenous infusion of DDMS (1 mg. kg(-1). h(-1)) or EETs and 20-HETE with 1-aminobenzotriazole (ABT; 2.2 mg. kg(-1). h(-1)) attenuated the ANG II-induced rise in MAP by 40%. Control urinary excretion of 20-HETE averaged 350 +/- 23 ng/day and increased to 1,020 +/- 105 ng/day in rats infused with ANG II (50 ng. kg(-1). min(-1)) for 5 days. In contrast, urinary excretion of 20-HETE only rose to 400 +/- 40 and 600 +/- 25 ng/day in rats chronically treated with ANG II and ABT or DDMS respectively. These results suggest that acute and chronic elevations in circulating ANG II levels increase the formation of 20-HETE in the kidney and peripheral vasculature and that 20-HETE contributes to the acute and chronic pressor effects of ANG II.     

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.     

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega-hydroxylase activity

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 +/- 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

Activation of Na+/H+ Exchanger NHE3 by Angiotensin II Is Mediated by Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3 (IRBIT) and Ca2+/Calmodulin-dependent Protein Kinase II

Angiotensin II (ANG II) stimulates renal tubular reabsorption of NaCl by targeting Na⁺/H⁺ exchanger NHE3. We have shown previously that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) plays a critical role in stimulation of NHE3 in response to elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i). In this study, we investigated the role of IRBIT in mediating NHE3 activation by ANG II. IRBIT is abundantly expressed in the proximal tubules where NHE3 is located. ANG II at physiological concentrations stimulates NHE3 transport activity in a model proximal tubule cell line. ANG II-induced activation of NHE3 was abrogated by knockdown of IRBIT, whereas overexpression of IRBIT enhanced the effect of ANG II on NHE3. ANG II transiently increased binding of IRBIT to NHE3 at 5 min but became dissociated by 45 min. In comparison, it took at least 15 min of ANG II treatment for an increase in NHE3 activity and NHE3 surface expression. The stimulation of NHE3 by ANG II was dependent on changes in [Ca²⁺]_i and Ca²⁺/calmodulin-dependent protein kinases II. Inhibition of CaMKII completely blocked the ANG II-induced binding of IRBIT to NHE3 and the increase in NHE3 surface abundance. Several serine residues of IRBIT are thought to be important for IRBIT binding. Mutations of Ser-68, Ser-71, and Ser-74 of IRBIT decreased binding of IRBIT to NHE3 and its effect on NHE3 activity. In conclusion, our current findings demonstrate that IRBIT is critically involved in mediating activation of NHE3 by ANG II via a Ca²⁺/calmodulin-dependent protein kinases II-dependent pathway.     

Effects of ANG II and III and angiotensin receptor blockers on nasal salt gland secretion and arterial blood pressure in conscious Pekin ducks (Anas platyrhynchos)

The vertebrate renin-angiotensin system controls cardiovascular, renal and osmoregulatory functions. Angiotensin II (ANG II) is the most potent hormone of the RAS but in some vertebrate animals angiotensin III (Val⁴-ANG III) may be a hormone. We studied the effects of some angiotensins and mammalian ANG II receptor antagonists on nasal salt gland function and arterial blood pressure in conscious white Pekin ducks. Nasal salt gland fluid secretion (NFS) was induced by a 10 ml · kg⁻¹ bw i.v. injection of a NaCl solution (1000 mosmol · kg⁻¹ H₂O) and maintained by a continuous i.v. infusion of the same solution at a rate of 0.97 ml · min⁻¹. There was a positive linear correlation between nasal fluid [Na⁺] and osmolality, between [Na⁺] and [K⁺], and also between the rate of NFS and [Na⁺] and [K⁺]. [Asp¹,Val⁵]-ANG II (1 nmol · kg⁻¹ i.v.) inhibited NFS but did not change ionic concentrations. Val⁴-ANG III (1 or 5 nmol · kg⁻¹) and ANG I (1-7) (20 nmol · kg⁻¹) had no effect on NFS. [Sar¹, Ile⁸]-ANG II (SARILE) acted as an ANG II receptor agonist and resulted in a prolonged and complete inhibition of NFS. The AT₁ receptor antagonist, losartan (DuP 753) and the AT₂ receptor antagonist, PD 123319 both failed to block the inhibitory effect of [Asp¹, Val⁵]-ANG II on the nasal salt glands. [Asp¹,Val⁵]-ANG II (2 nmol · kg⁻¹ i.v.) increased mean arterial blood pressure (MABP), whereas the same dose of [Asn¹,Val⁵]-ANG II (teleost) had only 30% of the pressor potency of the avian ANG II. Neither 1 nor 5 nmol · kg⁻¹ of Val⁴-ANG III i.v. nor 20 nmol · kg⁻¹ of ANG I (1-7) had any measurable effect on MABP. SARILE blocked completely the pressor response to [Asp¹,Val⁵]-ANG II but the AT₁ antagonists losartan and CGP 48933 and the AT₂ antagonist PD 123319 all failed to block the pressor response to [Asp¹,Val⁵]-ANG II. These results have substantiated an important role of the nasal salt gland in potassium regulation and highlighted a pharmacological dimorphism of saralasin, namely agonist and antagonist to angiotensin II-mediated inhibition of nasal salt gland function and pressor response, respectively. Using specific nonpeptidergic angiotensin II receptor antagonists, we have confirmed the distinct pharmacology of the avian angiotensin II receptors in a nongallinaceous species and the absence of significant angiotensin I (1-7) and angiotensin II effects on the cardiovascular system and nasal salt gland. Accepted: 6 November 1997     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Role of angiotensin II and its receptors in the development of fever in euhydrated and dehydrated rodents

(1) There are two types of angiotensin II (ANG II) receptors, AT₁ and AT₂. (2) In this paper, our starting point is our previous finding that hypothalamic AT₂-receptors modulate PGE-induced fever in a positive fashion. (3) We also present our recent results suggesting that ANG II, acting either peripherally or centrally, or both, contributes to the bacterial endotoxin-induced production of pyrogenic cytokines. (4) Taken together, our data suggest that, in the development of fever, hypothalamic ANG II and AT₂-receptors may be involved in the final step, and that ANG II also participates in the first step (namely, the bacterial endotoxin-induced synthesis of pyrogenic cytokines).     

Oscillatory Cl current induced by angiotensin II in rat pulmonary arterial myocytes: Ca dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca²⁺]_i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca²⁺]_i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (E_rev) of these currents was close to the equilibrium potential for Cl⁻ ions (E_Cl = −2.1 mV) and was shifted towards more positive values in low-Cl⁻ solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca²⁺]_i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca²⁺]_i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca²⁺]_i Niflumic acid (25 μM) had no effect on agonist-induced [Ca²⁺]_i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca²⁺]_i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca²⁺]_i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca²⁺]_i and that both oscillatory phenomena are primarily IP₃ mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca²⁺ channels. The resulting Ca²⁺ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

20-HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat

This study examined the effects of blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) on the acute fall in cerebral blood flow after subarachnoid hemorrhage (SAH) in the rat. In vehicle-treated rats, regional cerebral blood flow (rCBF) measured with laser-Doppler flowmetry fell by 30% 10 min after the injection of 0.3 ml of arterial blood into the cisterna magna, and it remained at this level for 2 h. Pretreatment with inhibitors of the formation of 20-HETE, 17-octadecynoic acid (17-ODYA; 1.5 nmol intrathecally) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 mg/kg iv), reduced the initial fall in rCBF by 40%, and rCBF fully recovered 1 h after induction of SAH. The concentration of 20-HETE in the cerebrospinal fluid rose from 12 +/- 2 to 199 +/- 17 ng/ml after SAH in vehicle-treated rats. 20-HETE levels averaged only 15 +/- 11 and 39 +/- 13 ng/ml in rats pretreated with 17-ODYA or HET0016, respectively. HET0016 selectively inhibited the formation of 20-HETE in rat renal microsomes with an IC(50) of <15 nM and human recombinant CYP4A11, CYP4F2, and CYP4F3 enzymes with an IC(50) of 42, 125, and 100 nM, respectively. These results indicate that 20-HETE contributes to the acute fall in rCBF after SAH in rats.     

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells

The accumulation of radiolabeled arachidonicacid (AA), immunoblot analysis of subcellular fractions, andimmunofluorescence tagging of proteins in intact cells were used toexamine the coupling of ANG II receptors with the activity and locationof a cytosolic phospholipase A₂(cPLA₂) in vascular smoothmuscle cells (VSMC). ANG II induced the accumulation of AA, whichpeaked by 10 min and was downregulated by 20 min. A large proportion ofthe AA released in response to ANG II was due to the activation of a Ca²⁺-dependent lipase coupled toan AT₁ receptor. However,regulation of Ca²⁺ availabilityfailed to completely block AA release, and a small but significantreduction in ANG II-mediated AA release was observed in the presence ofan AT₂ antagonist. These findings,coupled with a 25% reduction in the ANG II-induced AA release by aninhibitor specific for aCa²⁺-independentPLA₂, are consistent with thepresence and activation of aCa²⁺-independentPLA₂. In contrast, immunoblotanalysis and immunofluorescence detection showed that the ANGII-mediated translocation of cPLA₂ to a membrane fraction was exclusivelyAT₁ dependent and regulated byCa²⁺ availability. Furthermore,the nucleus was the membrane target. We conclude that ANG II regulatesthe Ca²⁺-dependent activation andtranslocation of cPLA₂ through anAT₁ receptor and that this eventis targeted at the nucleus in VSMC.

     

Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries

Background

Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.

Methodology/Principal Findings

Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K⁺ channel blockade with glibenclamide (K_ATP), 4-aminopyradine (K_V), BaCl₂ (K_IR) or tetraethylammonium (TEA, K_Ca) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca²⁺ fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca²⁺ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca²⁺ responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.

Conclusion

In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca²⁺ responses, independent of muscarinic receptor, NO or K⁺ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.     

Large conductance, Ca-activated K channels (BKCa) and arteriolar myogenic signaling

Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this vascular smooth muscle (VSM) response are events including membrane depolarization, Ca²⁺ entry and mobilization, and activation of contractile proteins. Large conductance, Ca²⁺-activated K⁺ channel (BK_Ca) has been implicated in several of these steps including, (1) channel closure causing membrane depolarization, and (2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BK_Ca activity (subunit composition, membrane potential (Em) and Ca²⁺ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity in BK_Ca channel activity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BK_Ca channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK_Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK_Ca in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK_Ca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NP_o) of BK_Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NP_o of BK_Ca channels, suggesting that the inhibitory effect of apelin-13 on BK_Ca is not mediated by a direct action on BK_Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK_Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK_Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.     

Effect of caveolin-1 scaffolding peptide and 17 -estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER) or ERβ. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17β-estradiol. One important system related to the activity of ER and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82–101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ER and ANG II type 1 and 2 (AT₁ and AT₂) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca²⁺ kinetics and the effect of 17β-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca²⁺ concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca²⁺ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT₂ receptors associate with Cav-1. Our results show that there is a close association of AT₁, AT₂, and ER with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling. estrogen receptors; angiotensin type 1 and 2 receptors; phosphatidylinositol 3-kinase; intracellular signaling; tissue culture; angiotensin receptors