Losartan counteracts the effects of cardiomyocyte swelling on glucose uptake and insulin receptor substrate-1 levels.

A growing body of evidence demonstrates an association between Angiotensin II (Ang II) receptor blockers (ARBs) and enhanced glucose metabolism during ischemic heart disease. Despite these encouraging results, the mechanisms responsible for these effects during ischemia remain poorly understood. In this study we investigated the influence of losartan, an AT1 receptor blocker, and secreted Ang II (sAng II) on glucose uptake and insulin receptor substrate (IRS-1) levels during cardiomyocyte swelling. H9c2 cells were differentiated to cardiac muscle and the levels of myogenin, Myosin Light Chain (MLC), and membrane AT1 receptors were measured using flow cytometry. Intracellular Ang II (iAng II) was overexpressed in differentiated cardiomyocytes and swelling was induced after incubation with hypotonic solution for 40min. Glucose uptake and IRS-1 levels were monitored by flow cytometry using 2-NBDG fluorescent glucose (10µM) or an anti-IRS-1 monoclonal antibody in the presence or absence of losartan (10-7M). Secreted Angiotensin II was quantified from the medium using a specific Ang II-EIA kit. To evaluate the relationship between sAng II and losartan effects on glucose uptake, transfected cells were pretreated with the drug for 24h and then exposed to hypotonic solution in the presence or absence of the secreted peptide. The results indicate that: (1) swelling of transfected cardiomyocytes decreased glucose uptake and induced the secretion of Ang II to the extracellular medium; (2) losartan antagonized the effects of swelling on glucose uptake and IRS-1 levels in transfected cardiomyocytes; (3) the effects of losartan on glucose uptake were observed during swelling only in the presence of sAng II in the culture medium. Our study demonstrates that both losartan and sAng II have essential roles in glucose metabolism during cardiomyocyte swelling.

Angiotensins and Huntington's Disease: A Study on Immortalized Progenitor Striatal Cell Lines.

Neurons from mouse models of Huntington's disease (HD) exhibit altered electrophysiological properties, potentially contributing to neuronal dysfunction and neurodegeneration. The renin-angiotensin system (RAS) is a potential contributor to the pathophysiology of neurodegenerative diseases. However, the role of angiotensin II (Ang II) and angiotensin (1-7) has not been characterized in HD. We investigated the influence of Ang II and angiotensin (1-7) on total potassium current using immortalized progenitor mutant huntingtin-expressing (Q111) and wild-type (Q7) cell lines. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results showed that (1) the effect of Ang II administered to the bath caused a negligible effect on potassium current in mutant Q111 cells compared with wild-type Q7 cells and that intracellular administration of Ang II reduced the potassium current in wild type but not in mutant cells; (2) the small effect of Ang II was abolished by losartan; (3) intracellular administration of Ang II performed in mutant huntingtin-expressing Q111 cells revealed a negligible effect of the peptide on potassium current; (4) flow cytometer analysis indicated a low expression of Ang II AT1 receptors in mutant Q111 cells; (5) mutant huntingtin-expressing striatal cells are highly sensitive to Ang (1-7) and that the effect of Ang (1-7) is related to the activation of Mas receptors. In conclusion, mutant huntingtin-expressing cells showed a negligible effect of Ang II on potassium current, a result probably due to the reduced expression of AT1 receptors at the surface cell membrane. In contrast, administration of Ang (1-7) to the bath showed a significant decline of the potassium current in mutant cells, an effect dependent on the activation of Mas receptors. Ang II had an intracrine effect in wild-type cells and Ang (1-7) exerted a significant effect in mutant huntingtin-expressing striatal cells.

Measurement of Cardiac Angiotensin II by Immunoassays, HPLC-Chip/Mass Spectrometry, and Functional Assays.

The molecular mechanisms related to the effect of angiotensin II, its level on cardiac tissues, as well as its overexpression represent an important aspect of cardiovascular pharmacology and pathology. Severe alterations of cardiac functions are induced by hypertension including activation of circulating and local cardiac renin angiotensin systems. In this chapter, we are providing the methods and materials necessary for further investigation of this important topic.

Local Renin Angiotensin Aldosterone Systems and Cardiovascular Diseases.

The presence of local renin angiotensin aldosterone systems (RAAS) in the cardiovascular and renal tissues and their influence in cardiovascular and renal diseases are described. The fundamental role of ACE/Ang II/AT1 receptor axis activation as well the counterregulatory role of ACE2/Ang (1-7)/Mas receptor activation on cardiovascular and renal physiology and pathology are emphasized. The presence of a local RAS and its influence on hypertension is discussed, and finally, the hypothesis that epigenetic factors change the RAAS in utero and induce the expression of renin or Ang II inside the cells of the cardiovascular system is presented.

Intracellular renin increases the inward calcium current in smooth muscle cells of mesenteric artery of SHR. Implications for hypertension and vascular remodeling.

UNLABELLED: The influence of intracellular renin on the inward calcium current in isolated smooth muscle cells from SHR mesenteric arteries was investigated. Measurements of calcium current were performed using the whole cell configuration of pCLAMP. The results indicated that: 1) renin (100nM) dialyzed into smooth muscle cells, increased the inward calcium current; 2) verapamil (10-9M) administered to the bath inhibited the effect of renin on the inward calcium current; 3) concurrently with the increase of calcium current a depolarization of 6.8+/-2.1mV (n=16)(P<0.05) was found in cells dialyzed with renin; 4) intracellular dialysis of renin (100nM) into smooth muscle cells isolated from mesenteric arteries of normal Wystar Kyoto rats showed no significant change on calcium current; 5) aliskiren (10-9M) dialyzed into the cell together with renin (100nM) abolished the effect of the enzyme on the calcium current in SHR; 6) Ang II (100nM) dialyzed into the smooth muscle cell from mesenteric artery of SHR in absence of renin, decreased the calcium current-an effect greatly reduced by valsartan (10-9M) added to the cytosol; 7) administration of renin (100nM) plus angiotensinogen (100nM) into the cytosol of muscles cells from SHR rats reduced the inward calcium current; 8) extracellular administration of Ang II (100nM) increased the inward calcium current in mesenteric arteries of SHR. CONCLUSIONS: intracellular renin in vascular resistance vessels from SHR due to internalization or expression, contributes to the regulation of vascular tone and control of peripheral resistance-an effect independently of Ang II. Implications for hypertension and vascular remodeling are discussed.

Exchange of chemical signals between cardiac cells. Fundamental role on cell communication and metabolic cooperation.

The exchange of chemical signals between cardiac cells and its relevance for cell communication and metabolic cooperation was reviewed. The role of gap junctions on the transfer of chemical information was discussed as well as the different factors involved in its regulation including changes in cell volume, high glucose, activation of the renin angiotensin aldosterone system including the intracrine effect of renin and angiotensin II on chemical coupling and cardiac energetics. Finally, the possible role of epigenetic changes of the renin angiotensin aldosterone system (RAAS) on the expression of components of the RAAS was discussed. The evidence available leads to the conception of the heart as a metabolic syncytium in which glucose as well nucleotides and hormones can flow from cell-to-cell though gap junctions, providing a new vision of how alterations in metabolic cooperation can induce cardiac diseases. These findings represent a stimulus for future research in this important area of cardiac physiology and pathology.

Intracellular angiotensin II as a regulator of muscle tone in vascular resistance vessels. Pathophysiological implications.

The influence of intracellular angiotensin II on the regulation of potassium current and membrane potential of smooth muscle cells of mesenteric arteries and its relevance for the regulation of vascular tone was reviewed. The presence of components of the renin angiotensin system (RAS) in different cells of the cardiovascular system, was discussed including their presence in the nuclei and mitochondria. Emphasis was given to the opposite effects of intracellular and extracellular angiotensin II (Ang II) on the regulation of potassium current, membrane potential and contractility of vascular resistance vessels and its implication to vascular physiology and pathology and the possible role of epigenetic factors on the expression of angiotensin II (Ang II) and renin in vascular resistance vessels as well as its possible pathophysiological role in hypertension and other cardiovascular diseases.

Aldosterone Disrupts the Intercellular Flow of Glucose in Cardiac Muscle.

The activation of the renin-angiotensin system is known to impair intercellular communication in the heart, but the role of aldosterone on the process of chemical communication and particularly the intercellular diffusion of glucose between cardiomyocytes is not known. This problem was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. For this, fluorescent glucose was dialyzed into one cell of the pair using the whole cell clamp technique, and its diffusion from cell-to-cell through gap junctions was followed by measuring the fluorescence intensity in the dialyzed as well as in non-dialyzed cell as a function of time. The results indicated that (1) in cell pairs exposed to aldosterone (100 nM) for 24 h, the intercellular flow of glucose through gap junctions was disrupted; (2) although the mechanism by which aldosterone disrupts the cell-to-cell flow of glucose is multifactorial, two major factors are involved: oxidative stress and PKC activation; (3) the effect of aldosterone was significantly reduced by spironolactone (100 nM); and (4) calculation of gap junction permeability (Pj) indicated an average values of 0.3 ± 0.001 × 10(-4) cm/s (n = 31) (four animals) for controls and 24 ± 0.03 × 10(-6) cm/s (n = 34) (four animals) (P < 0.05) for cell pairs exposed to aldosterone (100 nM) for 24 h. Bis-1 (10(-9)M), which is a selective PKC inhibitor, added to the aldosterone solution, improved the value of Pj to 0.21 ± 0.001 × 10(-4) cm/s (n = 24) (P < 0.05), whereas spironolactone (100 nM) added to aldosterone solution, reduced significantly the effect of the hormone on junctional permeability to glucose.

Chemical Communication between Heart Cells is Disrupted by Intracellular Renin and Angiotensin II: Implications for Heart Development and Disease.

HighlightsIntracellular renin and angiotensin disrupts chemical communication in heart.Epigenetic modification of renin angiotensin aldosterone system (RAAS) and heart disease.Intracrine renin angiotensin and metabolic cooperation.Gap junction, intracellular renin and angiotensin, cellular patterns, and heart development. The finding that intracellular renin and angiotensin II (Ang II) disrupts chemical communication and impairs metabolic cooperation between cardiomyocytes induced by aldosterone, hyperglycemia, and pathological conditions like myocardial ischemia is discussed. The hypothesis is presented that epigenetic changes of the renin angiotensin aldosterone system (RAAS) are responsible for cardiovascular abnormalities, including the expression of RAAS components inside cardiac myocytes (intracrine RAAS) with serious consequences including inhibition of electrical and chemical communication in the heart, resulting in metabolic disarrangement and cardiac arrhythmias. Moreover, the inhibition of gap junctional communication induced by intracellular Ang II or renin can contribute to the selection of cellular patterns during heart development.

Intracellular angiotensin (1-7) increases the inward calcium current in cardiomyocytes. On the role of PKA activation.

The influence of intracellular administration of angiotensin (1-7) (Ang 1-7) on the inward calcium current was investigated in myocytes isolated from the left ventricle of Wistar Kyoto rat hearts using the patch-clamp technique. The results indicated: (1) the intracellular administration of Ang (1-7) (100 nM) enhanced the peak inward calcium current (I Ca); (2) the intracellular administration of A779 (100 nM) which a Mas receptor inhibitor, abolished the effect of Ang (1-7) on the calcium current; (3) the activation of PKA and consequent phosphorylation of calcium channels seems to be the mechanism involved in the increment of calcium current induced by the heptapeptide because the intracellular dialysis of the PKA inhibitor suppressed the effect of the heptapeptide; (4) the effect of Ang (1-7) was not related to its secretion into the extracellular space; (5)intracellular dialysis of Ang II (100 nM) has an opposite effect and reduced the peak I Ca; (6) extracellular administration of Ang II (100 nM) to cells previously dialyzed with Ang (1-7) also reduced the peak I Ca previously enhanced by Ang (1-7); and (7) intracellular Ang (1-7) reduced the heart cell volume. Implications for heart contractility were discussed.

Intracellular angiotensin II disrupts chemical communication and impairs metabolic cooperation between cardiac myocytes.

The influence of intracellular angiotensin II (Ang II) on the process of chemical communication and metabolic cooperation between cardiac cells is discussed. Emphasis is given to the influence of pathological conditions like heart failure, myocardial ischemia or hyperglycemia on the activation of the intracrine renin angiotensin aldosterone system (RAAS) and its consequence for the metabolic cooperation between heart cells. Furthermore, the influence of high glucose on the process of chemical communication was described as well as its implication for the failing and diabetic heart. The major conclusion is that the activation of the intracrine renin angiotensin induced by heart failure, hyperglycemia, aldosterone or myocardial ischemia generates metabolic imbalance in the heart with serious consequences for the cardiac function.

Cell-to-cell diffusion of glucose in the mammalian heart is disrupted by high glucose. Implications for the diabetic heart.

The cell-to-cell diffusion of glucose in heart cell pairs isolated from the left ventricle of adult Wistar Kyoto rats was investigated. For this, fluorescent glucose was dialyzed into one cell of the pair using the whole cell clamp technique, and its diffusion from cell-to-cell was investigated by measuring the fluorescence in the dialyzed as well as in non-dialyzed cell as a function of time. The results indicated that: 1) glucose flows easily from cell-to-cell through gap junctions; 2) high glucose solution (25 mM) disrupted chemical communication between cardiac cells and abolished the intercellular diffusion of glucose; 3) the effect of high glucose solution on the cell-to-cell diffusion of glucose was drastically reduced by Bis-1 (10(-9)M) which is a PKC inhibitor; 4) intracellular dialysis of Ang II (100 nM) or increment of intracellular calcium concentration (10(-8)M) also inhibited the intercellular diffusion of glucose; 5) high glucose enhances oxidative stress in heart cells; 6) calculation of gap junction permeability (Pj) (cm/s) indicated a value of 0.74±0.08×10(-4) cm/s (5 animals) for the controls and 0.4±0.001×10(-5) cm/s; n=35 (5 animals) (P<0.05) for cells incubated with high glucose solution for 24h; 7) measurements of Pj for cell pairs treated with high glucose plus Bis-1 (10(-9)M) revealed no significant change of Pj (P>0.05); 8) increase of intracellular Ca(2+) concentration (10(-8)M) drastically decreased Pj (Pj=0.3±0.003×10(-5) cm/s). Conclusions indicate that: 1) glucose flows from cell-to-cell in the heart through gap junctions; 2) high glucose (25 mM) inhibited the intercellular diffusion of glucose-an effect significantly reduced by PKC inhibition; 3) high intracellular Ca(2+) concentration abolished the cell-to-cell diffusion of glucose; 4) intracellular Ang II (100 nM) inhibited the intercellular diffusion of glucose indicating that intracrine Ang II, in part activated by high glucose, severely impairs the exchange of glucose between cardiac myocytes. These observations support the view that the intracrine renin angiotensin system is a modulator of chemical communication in the heart. The implications of these findings for the diabetic heart were discussed.

Chemical communication between cardiac cells is disrupted by high glucose: implications for the diabetic heart.

UNLABELLED: The influence of high glucose solution on the chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. For this, Lucifer Yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique, and the diffusion of dye in the dialyzed as well as in non-dialyzed cell, was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that: 1) high glucose solution (25 mM) disrupted chemical communication between cardiac cells; 2) the effect of high glucose solution was reduced by Bis-1 (10(-9)M) which is a PKC inhibitor, and by enalapril (10(-9)M); 3) intracellular dialysis of Ang II (100 nM) also caused dye uncoupling; 4) calculation of gap junction permeability (Pj) (cm/s) indicated a value of 3 ± 0.07 × 10(-5)cm/s; n=32; (6 animals) for the controls and 0.4 ± 0.86 × 10(-6)cm/s; n=35 (6 animals) (P<0.05) for cells incubated with high glucose solution for 24h; 5) measurements of Pj for cell pairs treated with hypertonic solution plus Bis-1 (10(-9)M) or enalapril maleate (10(-9)M) showed no significant change of Pj (P>0.05). CONCLUSIONS: high glucose (25 mM) disrupts chemical communication between cardiac cells-an effect highly dependent on PKC activation. The possible role of enhanced intracellular Ang II levels induced by high glucose on the disruption of chemical communication was discussed as well as the possible implications of these findings for the diabetic heart.

Spironolactone enhances the beneficial effect of aliskiren on cardiac structural and electrical remodeling in TGR(mRen2)27 rats.

OBJECTIVE: To investigate the influence of simultaneous administration of spironolactone (20 mg/kg per day, intraperitoneal (i.p.)) and aliskiren (50 mg/kg per day, i.p.) for a period of eight weeks on cardiac remodeling in TGR(mRen2)27 rats. METHODS: Echocardiographic and electrophysiological and histological methods were used to determine the influence of spironolactone and aliskiren on cardiac remodeling. RESULTS: 1) the beneficial effect of aliskiren on SBP was enhanced by simultaneous administration of spironolactone; 2) echocardiographic studies showed that the left ventricle diameter (LVD), the left ventricle end diastolic volume (LVEDV) and the left ventricle posterior wall thickness (LVPW) were significantly reduced by the combination of both drugs when compared with aliskiren alone; 3) the ejection fraction was also increased; 4) histological studies indicated a greater decline in perivascular and interstitial fibrosis when both drugs were used; 5) the decrease of electrical remodeling of the left ventricle caused by aliskiren was further reduced by simultaneous administration of spironolactone; 6) the cardiac refractoriness increased by aliskiren was further incremented by spironolactone. Spironolactone (20 mg/kg per day) alone increased the ejection fraction and reduced LVD, LVEDV and LVPW but its effect was smaller than that achieved with the combination spironolactone plus aliskiren. CONCLUSION: The combination of an aldosterone inhibitor with a direct renin inhibitor proved to be of greater benefit for cardiac structural and electrical remodeling in this experimental model of hypertension than aliskiren alone.

Regulation of cell volume and water transport--an old fundamental role of the renin angiotensin aldosterone system components at the cellular level.

The expression and the role of renin angiotensin aldosterone system (RAAS) components on regulation of cell volume and water transport on vertebrates and invertebrates were reviewed. The presence of these components even in simple organisms like leeches and their relevance for the control of cellular volume and water transport supports the view that the expression of these components, at cellular level, is an acquisition which was preserved throughout evolution.

Clinical perspectives and fundamental aspects of local cardiovascular and renal Renin-Angiotensin systems.

Evidence for the potential role of organ specific cardiovascular renin-angiotensin systems (RAS) has been demonstrated experimentally and clinically with respect to certain cardiovascular and renal diseases. These findings have been supported by studies involving pharmacological inhibition during ischemic heart disease, myocardial infarction, cardiac failure; hypertension associated with left ventricular ischemia, myocardial fibrosis and left ventricular hypertrophy; structural and functional changes of the target organs associated with prolonged dietary salt excess; and intrarenal vascular disease associated with end-stage renal disease. Moreover, the severe structural and functional changes induced by these pathological conditions can be prevented and reversed by agents producing RAS inhibition (even when not necessarily coincident with alterations in arterial pressure). In this review, we discuss specific fundamental and clinical aspects and mechanisms related to the activation or inhibition of local RAS and their implications for cardiovascular and renal diseases. Fundamental aspects involving the role of angiotensins on cardiac and renal functions including the expression of RAS components in the heart and kidney and the controversial role of angiotensin-converting enzyme 2 on angiotensin peptide metabolism in humans, were discussed.

Angiotensin (1-7) re-establishes heart cell communication previously impaired by cell swelling: implications for myocardial ischemia.

UNLABELLED: The influence of hypertonic solution on dye coupling was investigated in cell pairs isolated from the left ventricle of adult Sprague Dawley rats.The hypertonic solution together with Lucifer Yellow CH, were dialyzed into one cell of the pair using the whole cell clamp tecnique, and the diffusion of dye in the dialyzed as well as in non-dialyzed cell, was followed by measuring the intensity of fluorescence in both cells as a function of time.The results indicated that: (1) Lucifer Yellow CH dialyzed into one cell of the pair diffuses easily into the nondialyzed cell through gap junctions; (2) the intracellular dialysis of an hypertonic solution into one cell of the pair, increases the area of the dialyzed cell and reduced the area of the non-dialyzed cell suggesting intercellular movement of water; (3) the hypertonic solution dialyzed into one cell of the pair abolished the dye coupling; (4) the gap junction permeability (Pj) estimated before and after administration of hypertonic solution showed an appreciably decrease of Pj; (5) angiotensin (1-7) (Ang (1-7) (10-9M) administered to the bath re-established the dye coupling abolished by hypertonic solution and reduced the cell area; (6) the effect of Ang (1-7) was related to the activation of Mas receptor and was dependent on the activation of PKA. CONCLUSIONS: the reestablishment of dye coupling elicited by Ang (1-7) seen in cell pairs dialyzed with hypertonic solution, might indicate that under similar conditions like that seen during myocardial ischemia, the peptide might be of benefit preventing the impairment of cell communication and impulse propagation associated with cardiac reentrant arrhytmias.

Angiotensin (1-7) increases the potassium current and the resting potential of arterial myocytes from vascular resistance vessels of normal adult rats: Pathophysiological implications.

The influence of angiotensin (Ang) (1-7) on potassium current (Kv) and resting potential of smooth muscle cells isolated from mesenteric artery of Sprague Dawley rats was investigated. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results indicated that Ang (1-7) (10(-9) M) increased the potassium current by 120% ± 2.6% (P < .05) and the resting potential of smooth muscle cells by 8 ± 2.8 mV (n = 23; P < .05). Ang II (10(-9) M) administered to the bath reduced the potassium current by 35% ± 3.6% (n = 23; P < .05) and depolarized the arterial myocytes by 7.8 ± 2.1 mV (n = 25; P < .05). The effect of the heptapeptide on potassium current was inhibited by a Mas receptor inhibitor (A779; 10(-8) M) as well as by a protein kinase A (PKA) inhibitor (10(-9) M) dialyzed into the cell. Intracellular dialysis of the catalytic subunit of PKA (5 × 10(-8) M) enhanced the potassium current by 38% ± 3.4% (n = 14; P < .05) but did not abolish the effect of Ang (1-7). On the other hand, Bis-1 (10(-9) M), which is a specific inhibitor of PKC, suppressed the effect of Ang (1-7) on potassium current. In conclusion, Ang (1-7) counteracts the effect of Ang II on potassium current and membrane potential of smooth muscle cells from mesenteric arteries, which are resistance vessels involved in the regulation of peripheral resistance and blood pressure. The activation of the cAMP/PKA cascade is essential for the effect of the heptapeptide. Pathophysiological implications are discussed.

Intracellular Renin Disrupts Chemical Communication between Heart Cells. Pathophysiological Implications.

HighlightsIntracellular renin disrupts chemical communication in the heartAngiotensinogen enhances the effect of reninIntracellular enalaprilat reduces significantly the effect of reninIntracellular renin increases the inward calcium currentHarmful versus beneficial effect during myocardial infarction The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; (1) under normal conditions, Lucifer Yellow flows from cell to cell through gap junctions; (2) the intracellular dialysis of renin (100 nM) disrupts chemical communication - an effect enhanced by simultaneous administration of angiotensinogen (100 nM); (3) enalaprilat (10(-9) M) administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; (4) aliskiren (10(-8) M) inhibited the effect of renin on chemical communication; (5) the possible role of intracellular renin independently of angiotensin II (Ang II) was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; (6) the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed; (7) the present results indicate that intracellular renin due to internalization or in situ synthesis causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.