High sucrose intake during gestation increases angiotensin II type 1 receptor-mediated vascular contractility associated with epigenetic alterations in aged offspring rats

Accruing evidence have confirmed that the fetal programming in response to adverse environmental in utero factors plays essential roles in the pathogenesis of hypertension in later life. High sugar intake has been accepted worldwide in everyday life diet and becomes the critical public health issue. Our previous studies indicated that intake of high sucrose (HS) during pregnancy could change the vascular reactivity and dipsogenic behavior closely associated with abnormal renin-angiotensin system (RAS), to increase the risk of hypertension in adult offspring. In the present study, we tested the hypothesis that maternal HS intake in pregnancy may further deteriorate the Ang II-induced cardiovascular responses in the aged offspring. HS intake was provided to pregnant rats throughout the gestation. Blood pressure (BP) in conscious state and vascular contractility in vitro were measured in 22-month-old aged offspring rats. In addition, mRNA and protein expressions and epigenetic changes of Ang II type 1 receptor (AT₁R) gene in blood vessels were determined with the methods of real-time RT-PCR, Western blotting, and Chromatin Immunoprecipitation Assay (CHIP). Results showed that, in the aged offspring, maternal HS intake during gestation would cause the elevation of basal BP which could be diminished by losartan. Although the circulatory Ang II was not changed, levels of local Ang II were significantly increased in blood vessels. In addition, prenatal HS exposure would significantly enhance the AT₁R-mediated vasoconstrictions in both aorta and mesenteric arteries of the aged offspring. Moreover, in the aged offspring of prenatal HS exposure, mRNA and protein expressions of AT₁R gene in both large and small blood vessels were significantly increased, which should be closely associated with the changes of epigenetic mechanisms such as histone modifications. Collectively, we proposed that maternal HS intake during gestation would cause abnormal BP responses mediated via the enhancement of vascular RAS, together with the increased expression of AT₁R gene related to the its epigenetic changes, which would actually lead to the overt phenotype of hypertension in the aged offspring.     

Central angiotensin II stimulates cutaneous water intake behavior via an angiotensin II type-1 receptor pathway in the Japanese tree frog Hyla japonica

Angiotensin II (Ang II) stimulates oral water intake by causing thirst in all terrestrial vertebrates except anurans. Anuran amphibians do not drink orally but absorb water osmotically through ventral skin. In this study, we examined the role of Ang II on the regulation of water-absorption behavior in the Japanese tree frog (Hyla japonica). In fully hydrated frogs, intracerebroventricular (ICV) and intralymphatic sac (ILS) injection of Ang II significantly extended the residence time of water in a dose-dependent manner. Ang II-dependent water uptake was inhibited by ICV pretreatment with an angiotensin II type-1 (AT₁) receptor antagonist but not a type-2 (AT₂) receptor antagonist. These results suggest that Ang II stimulates water-absorption behavior in the tree frog via an AT₁-like but not AT₂-like receptor. We then cloned and characterized cDNA of the tree frog AT₁ receptor from the brain. The tree frog AT₁ receptor cDNA encodes a 361 amino acid residue protein, which is 87% identical to the toad (Bufo marinus) AT₁ receptor and exhibits the functional characteristics of an Ang II receptor. AT₁ receptor mRNAs were found to be present in a number of tissues including brain (especially in the diencephalon), lung, large intestine, kidney and ventral pelvic skin. When tree frogs were exposed to dehydrating conditions, AT₁ receptor mRNA significantly increased in the diencephalon and the rhombencephalon. These data suggest that central Ang II may control water intake behavior via an AT₁ receptor on the diencephalon and rhombencephalon in anuran amphibians and may have implications for water consumption in vertebrates.     

Perinatal Na(+) Overload Programs Raised Renal Proximal Na(+) Transport and Enalapril-Sensitive Alterations of Ang II Signaling Pathways during Adulthood

Background

High Na⁺ intake is a reality in nowadays and is frequently accompanied by renal and cardiovascular alterations. In this study, renal mechanisms underlying perinatal Na⁺ overload-programmed alterations in Na⁺ transporters and the renin/angiotensin system (RAS) were investigated, together with effects of short-term treatment with enalapril in terms of reprogramming molecular alterations in kidney.

Methodology/Principal Findings

Male adult Wistar rats were obtained from dams maintained throughout pregnancy and lactation on a standard diet and drinking water (control) or 0.17 M NaCl (saline group). Enalapril (100 mg/l), an angiotensin converting enzyme inhibitor, was administered for three weeks after weaning. Ninety day old offspring from dams that drank saline presented with proximal tubules exhibiting increased (Na⁺+K⁺)ATPase expression and activity. Ouabain-insensitive Na⁺-ATPase activity remained unchanged but its response to angiotensin II (Ang II) was lost. PKC, PKA, renal thiobarbituric acid reactive substances (TBARS), macrophage infiltration and collagen deposition markedly increased, and AT₂ receptor expression decreased while AT₁ expression was unaltered. Early treatment with enalapril reduced expression and activity of (Na⁺+K⁺)ATPase, partially recovered the response of Na⁺-ATPase to Ang II, and reduced PKC and PKA activities independently of whether offspring were exposed to high perinatal Na⁺ or not. In addition, treatment with enalapril per se reduced AT₂ receptor expression, and increased TBARS, macrophage infiltration and collagen deposition. The perinatally Na⁺-overloaded offspring presented high numbers of Ang II-positive cortical cells, and significantly lower circulating Ang I, indicating that programming/reprogramming impacted systemic and local RAS.

Conclusions/Significance

Maternal Na⁺ overload programmed alterations in renal Na⁺ transporters and in its regulation, as well as severe structural lesions in adult offspring. Enalapril was beneficial predominantly through its influence on Na⁺ pumping activities in adult offspring. However, side effects including down-regulation of PKA, PKC and AT₂ receptors and increased TBARS could impair renal function in later life.     

Calreticulin enhances B2 bradykinin receptor maturation and heterodimerization

In different native tissues and cells the receptor for the vasodepressor bradykinin, B₂, forms dimers with the receptor for the vasopressor angiotensin II, AT₁. Because AT₁/B₂ heterodimers may contribute to enhanced angiotensin II-stimulated signaling under pathophysiological conditions, we analyzed mechanisms of AT₁/B₂ heterodimerization. We found that efficient B₂ receptor maturation was a prerequisite for heterodimerization because only the fully mature B₂ receptor was capable to interact with AT₁. To identify chaperones involved in B₂ receptor maturation and heterodimerization we performed microarray gene expression profiling of human embryonic kidney (HEK293) cells. The expression of the chaperone calreticulin was up-regulated in cells with efficient B₂ receptor maturation. Vice versa, upon down regulation of calreticulin expression by RNA interference, B₂ receptor maturation and AT₁/B₂ receptor heterodimerization were significantly impaired. Concomitantly, the B₂ receptor-mediated enhancement of AT₁-stimulated signaling was reduced. Thus, calreticulin enhances B₂ receptor maturation and heterodimerization with AT₁.     

Hippocampal apoptosis involved in learning deficits in the offspring exposed to maternal high sucrose diets

The hippocampus plays a crucial role in learning and memory, and neuronal apoptosis in the hippocampus contributes to learning deficits. Metabolism problems in pregnancy related to excessive fuel consumption (e.g., high fat, high sugar) may influence cognitive and behavioral functions in the offspring by affecting developing brain cells. This study determined the influence of maternal high sucrose (HS) diets on behavior and hippocampal neurons in the young offspring. The ratio of brain weight to body weight in the offspring exposed to prenatal HS diets was significantly decreased; the Morris water maze showed that the offspring exposed to prenatal HS diets exhibited increased escape latencies and path length during navigation testing, while there were no changes in time spent in the target quadrant and number of target approaches. In the offspring exposed to prenatal HS, TUNEL-positive cells were significantly increased in CA1, CA2 and CA3 of the hippocampus; protein expression of insulin-like growth factor-I, PI3K and phosphorylated Akt was significantly decreased, while caspase-3 and N-methyl-d-aspartate receptors were significantly increased in the hippocampus, and there was no change in expression of Bcl-2 and Akt. The results demonstrated that prenatal HS diets could induce the spatial acquisition deficits in the young offspring associated with hippocampal apoptosis, and altered signaling factors for antiapoptosis in the hippocampus might play a critical role in cognition disorders in young children.     

Reciprocal regulation of cholesterol excretion in apolipoprotein E-null mice by angiotensin II type 1 and type 2 receptor deficiency

AimsThe effects of AT₁ and AT₂ receptor deficiency on the intake and excretion of cholesterol were examined using atherosclerotic apolipoprotein E-null (ApoEKO) mice.Main methodsApoEKO, AT₁a/ApoEKO and AT₂/ApoEKO mice received a high-cholesterol diet (HCD: 1.25% cholesterol) for 10 days before sampling.Key findingsPlasma total cholesterol level was lower in AT₁a/ApoEKO mice and higher in AT₂/ApoEKO mice than in ApoEKO mice with a high cholesterol intake. In these mice, cholesterol content in feces was higher in AT₁a/ApoEKO mice and lower in AT₂/ApoEKO mice than in ApoEKO mice. Moreover, cholesterol content in bile tended to be higher in AT₁a/ApoEKO mice and lower in AT₂/ApoEKO mice than in ApoEKO mice, while a significant difference was observed only between AT₁a/ApoEKO and AT₂/ApoEKO mice. Cholesterol content and expression of HMG-CoA reductase and LDL receptor in liver were not different among the groups. Similar but weaker changes were also observed with a normal standard diet. Treatment with an AT₁ receptor blocker, irbesartan, increased cholesterol content in bile and tended to increase cholesterol excretion into feces in ApoEKO mice with HCD.SignificanceThese results suggest that AT₁ and AT₂ receptor stimulation was involved in the regulation of cholesterol excretion into bile and feces, and that the regulation acted reciprocally in a cholesterol overload condition with HCD.     

Different responses of atrial natriuretic peptide secretion and its receptor density to salt intake in rats

This study investigated whether high-salt intake influences atrial natriuretic peptide (ANP) system, atrial content, and release rate of ANP as well as receptor density in the kidney were measured in salt intake rats. Male Sprague-Dawley rats received either 0.9% or 2% salt in their drinking water for 10 days. The stretch-induced ANP secretion from isolated perfused non-beating left atria was accentuated, and the production of cGMP by ANP in renal cortical tissue membranes were pronounced in rats exposed to 0.9% salt for 10 days but not in rats exposed to 2% salt. The levels of ANP receptor density and expression in renal cortex were decreased in 2% salt intake rats but not in 0.9% salt intake rats. No significant differences in atrial and plasma concentrations of ANP and water balance were observed in both salt intakes. Therefore, these results suggest that atrial ANP secretion and its binding sites in the kidney may respond differently to ingested salt concentrations in rats.     

The ontogeny of components of the renin–angiotensin system in the porcine fetal ovary

There is an autonomous renin–angiotensin system (RAS) in the adult ovary. Renin is present in the primitive kidney, and the fetal ovary develops from the nephrogenic ridge. We hypothesised that components of the ovarian RAS would be present from early gestation, with potential roles in ovarian development. We studied fetal pig ovaries from approximately day 45 (～0.39 gestation) to term and measured mRNA (RT-PCR) for prorenin, angiotensinogen and the angiotensin II (AngII) Type 1 and 2 receptors (AT₁ and AT₂), and protein expression (Western blot) and localization (immunohistochemistry) of the AT₁ and AT₂ receptors. mRNA for prorenin was present in relatively low abundance from at least day 45 and rose to ～day 75 of gestation, whilst mRNA for angiotensinogen rose steadily. mRNA for the AT₁ receptor was present from approximately day 45 and did not alter significantly with increasing gestation but AT₂ receptor mRNA was initially high, falling sharply through pregnancy. The AT₁ receptor protein abundance fell steadily to term, whereas the AT₂ receptor protein did not change during gestation. Both receptors were localised in the surface epithelium and egg nests, the granulosa cells of primordial, primary and secondary follicles, and the oocytes of all except the secondary follicles. Collectively, our results support the hypothesis that there is a functional RAS in the fetal ovary from at least approximately day 45 of gestation until term and that it may have a paracrine role in ovarian growth and development.     

Dopamine and G protein-coupled receptor kinase 4 in the kidney: Role in blood pressure regulation

Complex interactions between genes and environment result in a sodium-induced elevation in blood pressure (salt sensitivity) and/or hypertension that lead to significant morbidity and mortality affecting up to 25% of the middle-aged adult population worldwide. Determining the etiology of genetic and/or environmentally-induced high blood pressure has been difficult because of the many interacting systems involved. Two main pathways have been implicated as principal determinants of blood pressure since they are located in the kidney (the key organ responsible for blood pressure regulation), and have profound effects on sodium balance: the dopaminergic and renin–angiotensin systems. These systems counteract or modulate each other, in concert with a host of intracellular second messenger pathways to regulate sodium and water balance. In particular, the G protein-coupled receptor kinase type 4 (GRK4) appears to play a key role in regulating dopaminergic-mediated natriuresis. Constitutively activated GRK4 gene variants (R65L, A142V, and A486V), by themselves or by their interaction with other genes involved in blood pressure regulation, are associated with essential hypertension and/or salt-sensitive hypertension in several ethnic groups. GRK4γ ?142V?transgenic mice are hypertensive on normal salt intake while GRK4γ? 486V? transgenic mice develop hypertension only with an increase in salt intake. GRK4 gene variants have been shown to hyperphosphorylate, desensitize, and internalize two members of the dopamine receptor family, the D₁ (D₁R) and D₃ (D₃R) dopamine receptors, but also increase the expression of a key receptor of the renin–angiotensin system, the angiotensin type 1 receptor (AT₁R). Knowledge of the numerous blood pressure regulatory pathways involving angiotensin and dopamine may provide new therapeutic approaches to the pharmacological regulation of sodium excretion and ultimately blood pressure control.     

Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

Previous studies have reported that perinatal nicotine exposure causes development of hypertensive phenotype in adult offspring.

Aims

The present study was to determine whether perinatal nicotine exposure causes an epigenetic programming of vascular Angiotensin II receptors (ATRs) and their-mediated signaling pathway leading to heightened vascular contraction in adult offspring.

Main methods

Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth. The experiments were conducted at 5 months of age of male offspring.

Key Findings

Nicotine treatment enhanced Angitension II (Ang II)-induced vasoconstriction and 20-kDa myosin light chain phosphorylation (MLC₂₀-P) levels. In addition, the ratio of Ang II-induced tension/MLC-P was also significantly increased in nicotine-treated group compared with the saline group. Nicotine-mediated enhanced constrictions were not directly dependent on the changes of [Ca²⁺]_i concentrations but dependent on Ca²⁺ sensitivity. Perinatal nicotine treatment significantly enhanced vascular ATR type 1a (AT_1aR) but not AT_1bR mRNA levels in adult rat offspring, which was associated with selective decreases in DNA methylation at AT_1aR promoter. Contrast to the effect on AT_1aR, nicotine decreased the mRNA levels of vascular AT₂R gene, which was associated with selective increases in DNA methylation at AT₂R promoter.

Significance

Our results indicated that perinatal nicotine exposure caused an epigenetic programming of vascular ATRs and their-mediated signaling pathways, and suggested that differential regulation of AT₁R/AT₂R gene expression through DNA methylation mechanism may be involved in nicotine-induced heightened vasoconstriction and development of hypertensive phenotype in adulthood.     

Dietary sodium manipulation during critical periods in development sensitize adult offspring to amphetamines

This study examined critical periods in development to determine when offspring were most susceptible to dietary sodium manipulation leading to amphetamine sensitization. Wistar dams (n = 6-8/group) were fed chow containing low (0.12% NaCl; LN), normal (1% NaCl; NN), or high sodium (4% NaCl; HN) during the prenatal or early postnatal period (birth to 5 wk). Offspring were fed normal chow thereafter until testing at 6 mo. Body weight (BW), blood pressure (BP), fluid intake, salt preference, response to amphetamine, open field behavior, plasma adrenocorticotropin hormone (ACTH), plasma corticosterone (Cort), and adrenal gland weight were measured. BW was similar for all offspring. Offspring from the prenatal and postnatal HN group had increased BP, NaCl intake, and salt preference and decreased water intake relative to NN offspring. Prenatal HN offspring had greater BP than postnatal HN offspring. In response to amphetamine, both prenatal and postnatal LN and HN offspring had increased locomotor behavior compared with NN offspring. In a novel open field environment, locomotion was also increased in prenatal and postnatal LN and HN offspring compared with NN offspring. ACTH and Cort levels 30 min after restraint stress and adrenal gland weight measurement were greater in LN and HN offspring compared with NN offspring. These results indicate that early life experience with low- and high-sodium diets, during the prenatal or early postnatal period, is a stress that produces long-term changes in responsiveness to amphetamines and to subsequent stressors.     

Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4')

Although angiotensin II (Ang II) binds to Ang II type 1 (AT₁) and type 2 (AT₂) receptors, AT₁ and AT₂ receptors have antagonistic actions with regard to cell signaling. The molecular mechanisms that underlie this antagonism are not well understood. We examined AT₁ and AT₂ receptor-induced signal cross-talk in the cytoplasm and the importance of the hetero-dimerization of AT₁ receptor with AT₂ receptor on the cell surface. AT₁ and AT₂ receptors showed antagonistic effects toward inositol phosphate production. AT₁ receptors mainly formed homo-dimers, rather than hetero-dimers with AT₂ receptor, on the cell surface as determined by immunoprecipitation, and subsequently induced cell signals. AT₂ receptor mainly formed homo-dimers, rather than hetero-dimers with AT₁ receptor, on the cell surface. The expression levels of homo-dimerized AT₁ receptor or AT₂ receptor on the cell surface did not change after treatment with Ang II, the AT₁ receptor antagonist telmisartan or the AT₂ receptor antagonist PD123319. Finally, AT₁ and AT₂ receptor-induced signals antagonized phospholipase C-β₃ phosphorylation. In conclusion, Ang II-induced AT₁ receptor signals may be mainly blocked by AT₂ receptor signals through their negative cross-talk in the cytoplasm rather than by the hetero-dimerization of both receptors on the cell surface. The proper balance of the expression levels of AT₁ and AT₂ receptors might be critical for the antagonistic action between these receptors.     

Investigations on the physiological controls of water and saline intake in C57BL/6 mice

To examine the behavioral and neural control of body fluid homeostasis, water and saline intake of C57BL/6 mice was monitored under ad libitum conditions, after treatments that induce water or salt intake, and after ablation of the periventricular tissue of the anteroventral third ventricle (AV3V). Mice have nocturnal drinking that is most prevalent after the offset and before the onset of lights. When given ad libitum choice, C57BL/6 mice show no preference for saline over water at concentrations up to 0.9% NaCl and a progressive aversion to saline above that concentration. Systemic hypertonic saline, isoproterenol, and polyethylene glycol treatments are dipsogenic; however, systemic ANG II is not. Intracerebroventricular injections of both hypertonic saline and ANG II are dipsogenic, and diuretic treatment followed by a short period of sodium deprivation induces salt intake. After ablation of the AV3V, mice can be nursed to recovery from initial adipsia and, similar to rats, show chronic deficits to dipsogenic treatments. Taken together, the data indicate that mechanisms controlling thirst in response to cellular dehydration in C57BL/6 mice are similar to rats, but there are differences in the efficacy of extracellular dehydration-related mechanisms, especially for systemic ANG II, controlling thirst and salt appetite.     

ONTOGENY OF ANGIOTENSIN II RECEPTORS

Aside from the well known role of angiotensin II (Ang II) in blood pressure regulation and fluid homeostasis, accumulating evidence suggests that the octapeptide hormone also plays a role in growth and development. There are two major classes of Ang II receptors (AT₁and AT₂) which mediate Ang II action. Both classes are members of the large superfamily of seven transmembrane domain spanning receptors. Fetal tissue express high levels of AT receptors. Throughout fetal and postpartum life, the AT₁and AT₂tissue distribution changes dramatically. The evolution of each receptor type is distinct and varies according to the organ. Thus, the different patterns of temporal expression of each receptor class could be related to various roles that Ang II may play during development.     

Down-regulation of Cyclooxygenase-2 by the Carboxyl Tail of the Angiotensin II Type 1 Receptor

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT₁) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT₁ also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT₁ enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT₁ in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT₁ may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.     

Angiotensin AT2 Receptor Contributes towards Gender Bias in Weight Gain

Obesity is a major disease condition, in turn leading to pathological changes collectively recognized as metabolic syndrome. Recently angiotensin receptor AT₂R has been associated negatively with body weight (BW) gain in male mice. However, the gender differences in AT₂R and BW changes have not been studied. To understand the gender based role of AT₂R involving BW changes, we fed male and female wild type (WT) and AT₂R knock out (AT₂KO) mice with C57BL6 background with high fat diet (HFD) for 16 weeks. The male AT₂KO had higher HFD calorie intake (WT: 1280±80; AT₂KO:1680±80 kcal) but gained less BW compared with the WT (WT: 13; AT₂KO: 6 g). Contrary to the male animals, the female AT₂KO mice with equivalent caloric intake (WT: 1424±48; AT₂KO:1456±80 kcal) gained significantly more BW than the WT mice (WT: 9 g; AT₂KO: 15 g). The male AT₂KO on HFD displayed lower plasma insulin level, less impaired glucose tolerance (GT), and higher plasma T3 compared with WT males on HFD; whereas the female AT₂KO mice on HFD showed elevated levels of plasma insulin, more impaired GT, lower plasma T3 and higher free fatty acid and hepatic triglycerides compared with WT females on HFD. Interestingly, compared with WT, AT₂KO female mice had significantly lower estrogen, which was further reduced by HFD. These results suggest that AT₂R in female mice via potentially regulating estrogen may have protective role against BW gain and impaired glucose tolerance and lipid metabolism.