Appraisal of the role of angiotensin II and aldosterone in ventricular myocyte apoptosis in adult normotensive rat

Despite previous observations on isolated ventricular myocytes, there are still few evidences that angiotensin II induces cardiomyocyte apoptosis in vivo. The possibility that aldosterone, the final hormone of the renin-angiotensin-aldosterone system under Ang II control, can stimulate cardiac apoptosis has not yet been explored. Angiotensin II or aldosterone (1mg/kg each) were infused in adult normotensive rats for different times, and the number of apoptotic ventricular myocyte nuclei was quantified by the TUNEL method, along with caspase-3 activation. The role of angiotensin II type 1 receptor in vivo was assessed by selective blockade with valsartan and ex vivo by binding experiments. In addition, myocytes in primary culture were incubated with Ang II or aldosterone in presence of spironolactone. Continuous infusion of Ang II induced a rapid, AT(1)-mediated increase of apoptotic cardiomyocyte nuclei (from 14+/-9 to 188+/-35 TdT-labeled nuclei/10(6) after 3h, P<0.005) and of activated caspase-3, that normalized after 24h. The normalization was associated with a down-regulation of myocardial AT(1) receptors. Aldosterone stimulated cardiomyocyte apoptosis both in vivo and in isolated cells, to a similar extent as Ang II. The maximal apoptotic rate reported here ( approximately 0.02%) and the transient effect of Ang II suggest that myocyte loss by apoptosis is limited in the present model. The data on aldosterone-induced ventricular myocyte apoptosis deserve further attention to delineate the role of aldosterone in cell death and offer possible mechanistic explanations on the benefits afforded by aldosterone receptor antagonists in heart failure.     

Hyperlipidemic mice present enhanced catabolism and higher mitochondrial ATP-sensitive K+ channel activity

BACKGROUND & AIMS: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. METHODS: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) agonists and antagonists were measured in isolated mitochondria, livers, and mice. RESULTS: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoK(ATP) agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoK(ATP). The higher content and activity of liver mitoK(ATP) resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body CO(2) release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. CONCLUSIONS: These results show that primary hyperlipidemia leads to an elevation in liver mitoK(ATP) activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoK(ATP), in addition to UCPs, may be involved in the control of energy metabolism and body weight.     

Aldosterone: Role in the Cardiometabolic Syndrome and Resistant Hypertension

The prevalence of diabetes, hypertension, and cardiovascular disease (CVD) and chronic kidney disease (CKD) is increasing in concert with obesity. Insulin resistance, metabolic dyslipidemia, central obesity, albuminuria. and hypertension commonly cluster to comprise the cardiometabolic syndrome (CMS). Emerging evidence supports a shift in our understanding of the crucial role of elevated serum aldosterone in promoting insulin resistance and resistant hypertension. Aldosterone enhances tissue generation of oxygen free radicals and systemic inflammation. This increase in oxidative stress and inflammation, in turn, contributes to impaired insulin metabolic signaling, reduced endothelial-mediated vasorelaxation, and associated cardiovascular and renal structural and functional abnormalities. In this context, recent investigation indicates that hyperaldosteronism, which is often associated with obesity, contributes to impaired pancreatic β-cell function as well as diminished skeletal muscle insulin metabolic signaling. Accumulating evidence indicates that the cardiovascular and renal abnormalities associated with insulin resistance are mediated, in part, by aldosterone's nongenomic as well as genomic signaling through the mineralocorticoid receptor (MR). In the CMS, there are increased circulating levels of glucocorticoids, which can also activate MR signaling in cardiovascular, adipose, skeletal muscle, neuronal, and liver tissue. Furthermore, there is increasing evidence that fat tissue produces a lipid soluble factor that stimulates aldosterone production from the adrenal zona glomerulosa. Recently, we have learned that MR blockade improves pancreatic insulin release, insulin-mediated glucose utilization, and endothelium-dependent vasorelaxation as well as reduces the progression of CVD and CKD. In summary, aldosterone excess exerts detrimental metabolic effects that contribute to the development of the CMS and resistant hypertension as well as CVD and CKD.     

Lipid rafts establish calcium waves in hepatocytes

BACKGROUND & AIMS: Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. METHODS: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. RESULTS: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. CONCLUSIONS: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.     

Interaction of calcium/calmodulin-dependent protein kinase IIdeltaC with sorcin indirectly modulates ryanodine receptor function in cardiac myocytes

Calcium/calmodulin dependent protein kinase II delta C (CaMKIIdelta(C)) and the EF-hand Ca(2+)-binding protein, sorcin have both been shown to regulate the excitation-contraction coupling process. This study explores the possibility that these two proteins interact directly and, as a result of this interaction, modulate cardiac calcium handling. Two independent methods (surface plasmon resonance (SPR) and overlay assays) were used to determine whether CaMKIIdelta(C) and sorcin interacted in a direct manner. The nature of this interaction was explored by (i) examining the effects of sorcin on CaMKIIdelta(C) activity using a selective kinase assay and (ii) studying whether sorcin was a substrate for CaMKIIdelta(C) using autoradiography. Ryanodine binding assays on mouse ventricular cardiomyocytes were used to determine specific functional effects of this interaction. SPR studies suggested that sorcin interacts with CaMKIIdelta(C) in a concentration-dependent manner. This interaction occurs in the presence of Ca(2+) and in the presence or absence of calmodulin (CaM). Overlay assays confirmed the existence of this interaction. Further experiments suggested that this interaction is reciprocal. Firstly, sorcin significantly inhibited both recombinant and native CaMKIIdelta(C) activity to similar extents. Secondly, sorcin was phosphorylated by CaMKIIdelta(C). Thirdly, sorcin inhibition of CaMKII activity occurred under conditions where sorcin remained dephosphorylated. Functionally, CaMKIIdelta(C)-mediated phosphorylation of sorcin served to abolish the inhibitory effect of sorcin on ryanodine receptor (RyR(2)) open probability (Po). Since both proteins are capable of directly modulating RyR(2) activity, this interaction may serve as an additional or alternative indirect route by which both proteins can regulate RyR(2) opening status in cardiac myocytes.     

Lysophosphatidylcholine enhances I(Ks) currents in cardiac myocytes through activation of G protein, PKC and Rho signaling pathways

Lysophosphatidylcholine (LPC) is a bioactive phospholipid that accumulates rapidly in the ischemic myocardium. In recent years, it has been shown that some of the actions of LPC are mediated through the activation of the membrane G proteins. However, the precise mechanism(s) responsible for the LPC-related intracellular signaling in the regulation of cardiac ion channels are still poorly understood. The present study was undertaken to examine whether LPC regulates the slow component of the delayed rectifier K⁺ current (I_Ks) and, if so, what intracellular signals are important for this process. Isolated guinea pig cardiac myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp method. The bath application of 1-palmitoyl-lysophosphatidylcholine (LPC-16) concentration-dependently (EC₅₀ = 0.7 μM) and reversibly increased I_Ks in atrial cells, but failed to potentiate I_Ks in ventricular myocytes. In contrast, 1-oleoyl-lysophosphatidylcholine (LPC-18:1) only produced a slight I_Ks increase, and 1-caproyl-lysophosphatidylcholine (LPC-6) or the LPC-16 precursor (phosphatidylcholine) had no effect on I_Ks. Pretreatment of atrial cells with an antibody against the N-terminus of the G2A receptor significantly reduced the LPC-16-induced potentiation of I_Ks. The inhibition of heterotrimeric G protein, phospholipase C (PLC) and protein kinase C (PKC) significantly reduced LPC-16-induced enhancement of I_Ks. Moreover, the blockade of Rho and Rho-kinase by specific inhibitors also inhibited the activity of LPC-16. Immunohistochemical studies demonstrated that G2A was densely distributed in the plasma membrane of atrial myocytes. Therefore, the present study suggests that the activation of a G protein (probably Gα_q) by LPC-16 potentiates I_Ks currents through the PLC-PKC and Rho-kinase pathways.     

Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis

BACKGROUND & AIMS: There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. METHODS: The presence of the delta opioid receptor (OR), muOR, and kappaOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. RESULTS: Cholangiocytes express all 3 receptors studied. deltaOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas muOR resulted in a slight increase in cell growth. The deltaOR signal is mediated by the IP3/CamKIIalpha/PKCalpha pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, muOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIalpha/PKCalpha pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. CONCLUSIONS: The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the deltaOR expressed by cholangiocytes.     

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes

β₁-Adrenergic receptors (β₁ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β₁ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β₁AR activation enhanced the L-type Ca²⁺ current, intracellular Ca²⁺ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β₁ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β₁AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β₁AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β₁AR and EPR responses. They also suggest that β₁AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.     

Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner

BACKGROUND & AIMS: Prevention of bile acid-induced apoptosis is of therapeutic interest and requires the understanding of underlying mechanisms. METHODS: The effect of tauroursodeoxycholate (TUDC) on taurolithocholic acid-3 sulfate (TLCS)-induced apoptosis was studied in cultured rat hepatocytes. RESULTS: TLCS induced activation of caspases 8, 9, and 3 and hepatocyte apoptosis. These effects were abolished by TUDC in a PI 3-kinase-/protein kinase B (PKB)-, p38(MAPK)-, and extracellular signal-regulated kinase-2 (Erk-2)-independent manner. These protein kinases were activated by both TLCS and TUDC, however, with different kinetics. TLCS, but not TUDC, led to a sustained activation of c-Jun N-terminal kinase (JNK) and CD95 trafficking to the plasma membrane; both TLCS effects were prevented by TUDC. Inhibition of JNK1 or protein kinase C prevented TLCS-induced CD95 membrane trafficking and blunted the apoptotic response. The apoptotic potency of other bile acids paralleled their ability to induce sustained JNK activation. CONCLUSIONS: Protection by TUDC against TLCS-induced apoptosis starts upstream of caspase 8 activation and is independent of a PI 3-kinase-dependent survival pathway. JNK activation may be important for bile acid-induced apoptosis by triggering ligand-independent CD95 surface trafficking and activation of apoptosis.     

Activation of natriuretic peptide receptor-C attenuates the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats: implication of Gialpha protein

We have recently shown that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of Giα proteins, which was attributed to the enhanced oxidative stress. Since C-ANP_4-23 that specifically interacts with natriuretic peptide C (NPR-C) receptor has been shown to decrease the expression of Giα protein in VSMC, the present study was undertaken to examine if C-ANP_4-23 can also decrease the enhanced expression of Giα protein in VSMC from SHR and whether it is attributed to its ability to attenuate the enhanced oxidative stress. Aortic VSMC from 12-week-old SHR and their age-matched Wistar-Kyoto (WKY) rats were used for the present studies. VSMC from SHR exhibited enhanced expression of Giα-2 and Giα-3 proteins, different subunits of NADPH oxidase such as Nox⁴ and p^47phox proteins but not of p^22phox, enhanced production of superoxide anion as well as NADPH oxidase activity as compared to age-matched WKY rats. Treatment of VSMC from SHR with C-ANP_4-23 decreased towards control levels the enhanced expression of Giα proteins, enhanced superoxide anion production and enhanced NADPH oxidase activity as well as the enhanced expression of Nox⁴ and p^47phox. However, C-ANP_4-23-induced attenuation of the enhanced level of O₂⁻ and NADPH oxidase activity occurs at 4 h before the decrease in the enhanced expression of p^47phox that occurs at 16 h of C-ANP_4-23 treatment. The decreased expression of NADPH oxidase in SHR was also associated with further decrease in O₂⁻ and NADPH oxidase activity. Furthermore, treatment of VSMC from SHR with pertussis toxin (PT) decreased the enhanced levels of superoxide anion as well as NADPH oxidase activity; however, the enhanced levels of different subunits of NADPH oxidase were not attenuated by PT treatment. These results suggest that C-ANP_4-23 decreases the enhanced oxidative stress in SHR by attenuating the enhanced expression of Giα proteins and also the enhanced levels of NADPH oxidase.     

Carbonic anhydrases and mucosal vanilloid receptors help mediate the hyperemic response to luminal CO2 in rat duodenum

BACKGROUND & AIMS: The duodenal mucosa is exposed to PCO(2) >200 mm Hg due to the luminal mixture of gastric acid with secreted bicarbonate, which augments mucosal protective mechanisms. We examined the hyperemic response to elevated luminal PCO(2) in the duodenum of anesthetized rats luminally exposed to high CO(2) saline to help elucidate luminal acid-sensing mechanisms. METHODS: Blood flow was measured by laser Doppler, and intracellular pH of epithelial cells by measured by ratio microimaging. The permeant carbonic anhydrase (CA) inhibitor methazolamide, relatively impermeant CA inhibitor benzolamide, vanilloid receptor antagonist capsazepine, or sodium-hydrogen exchanger 1 (NHE-1) inhibitor dimethyl amiloride were perfused with or without the high CO(2) solution. RESULTS: The high CO(2) solution increased duodenal blood flow, which was abolished by pretreatment with methazolamide or capsazepine or by dimethyl amiloride coperfusion. Sensory denervation with capsaicin also abolished the CO(2) effects. Benzolamide dose-dependently inhibited CO(2)-induced hyperemia and at 100 nmol/L inhibited CO(2)-induced intracellular acidification. The membrane-bound CA isoforms IV, IX, XII, and XIV and cytosolic CA II and the vanilloid receptor 1 (TRPV1) were expressed in duodenum and stomach. Dorsal root ganglion and nodose ganglion expressed all isoforms except for CA IX. Conclusions: The duodenal hyperemic response to luminal CO(2) is dependent on cytosolic and membrane-bound CA isoforms, NHE-1, and TRPV1. CO(2)-induced intracellular acidification was inhibited by selective extracellular CA inhibition, suggesting that CO(2) diffusion across the epithelial apical membrane is mediated by extracellular CA. NHE-1 activation preceding TRPV1 stimulation suggests that luminal CO(2) is sensed as H(+) in the subepithelium.     

Activation of estrogen receptor alpha disrupts differentiation of the reproductive organs in chicken embryos

Gonadal estrogen plays an important role in the differentiation of a female phenotype in birds. Exogenous compounds that interfere with estrogen signaling, for instance by binding to the estrogen receptors alpha and beta (ERα and ERβ), are therefore potential disruptors of sexual differentiation in birds. The ERα agonist propyl-pyrazole-triol (PPT), the ERα antagonist methyl piperidino pyrazole (MPP) and the ERβ agonist diarylproprionitrile (DPN) were used in the present study to explore the roles of the ERs in normal and disrupted sex differentiation in the chicken embryo. Activation of ERα by PPT caused disturbed differentiation of the reproductive organs in both sexes. In male embryos, PPT caused left-side ovotestis formation and retention of the Müllerian ducts. In female embryos, PPT caused retention of the right Müllerian duct (which normally regresses) and malformation of both Müllerian ducts. PPT also induced hepatic expression of mRNA for the estrogen-regulated egg yolk protein apoVLDL II. Notably, none of these effects were observed following treatment with DPN. ERα-inactivation by MPP counteracted the action of PPT but had little effect by its own. Our results indicate that ERα plays an important role in sex differentiation of the reproductive tract in female chicken embryos and show that ERα can mediate xenoestrogen-induced disturbances of sex differentiation.     

Molecular determinants of angiotensin II type 1 receptor functional selectivity

The angiotensin AT₁ receptor is an important pharmacological target in the treatment of cardiovascular disorders, such as hypertension, diabetic nephropathy, cardiac hypertrophy, arrhythmia and failure. Simultaneously, the AT₁ receptor has emerged to be a prominent model for the emerging concept that receptors may attain multiple active states with differentiated functional outcomes. Two major signalling pathways are employed by the AT₁ receptor, namely 1) the canonical G_q protein-dependent activation of inositol phosphate turnover and intracellular calcium release, and 2) G protein-independent recruitment of β-arrestin-scaffolded signalling complexes that activate protein kinase pathways. Different states of receptor activation with preference for individual downstream pathways (functional selectivity) have been demonstrated in mutational studies of the AT₁ receptor and by pharmacological probing with analogues of angiotensin II. These studies also provide clues about the conformational changes that underlie different functional outcomes. In this review, we evaluate current knowledge of the molecular determinants of AT₁ receptor activation, which may distinguish G protein-dependent and -independent behaviour. While G protein activation is known to be detrimental, G protein-independent signalling by the AT₁ receptor has been associated with phenotypes such as cell survival and renewal, regulation of cardiac contraction and cell migration. It is therefore currently hypothesized that selective blockade of G protein actions and simultaneous activation of G protein-independent signalling will prove to be a feasible strategy for improved cardiovascular therapy. The pharmacological perspectives of functional selectivity by receptors, such as the AT₁ receptor, urge the elucidation of molecular mechanisms that govern disparate signalling events.     

Air pollution and cardiovascular disease in the Multi-Ethnic Study of Atherosclerosis

Research to date demonstrates a relationship between exposure to ambient air pollutants and cardiovascular disease (CVD). Many studies have shown associations between short-term exposures to elevated levels of air pollutants and CVD events, and several cohort studies suggest effects of long-term exposure on cardiovascular mortality, coronary heart disease events, and stroke. The biologic mechanisms underlying this long-term exposure relationship are not entirely clear but are hypothesized to include systemic inflammation, autonomic nervous system imbalance, changes in vascular compliance, altered cardiac structure, and development of atherosclerosis. The Multi-Ethnic Study of Atherosclerosis provides an especially well-characterized population in which to investigate the relationship between air pollution and CVD and to explore these biologic pathways. This article reviews findings reported to date within this cohort and summarizes the aims and anticipated contributions of a major ancillary study, the Multi-Ethnic Study of Atherosclerosis and Air Pollution.