Volume 5, Number 4, December 2004

POPULAR
TOPICPAPERElevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II
Enzo R Porrello, Catherine E Huggins, Claire L Curl, Andrea A Domenighetti, Thierry Pedrazzini, Leanne MD Delbridge, Trefor O Morgan

Introduction/hypothesis: Cardiac hypertrophy is an independent risk factor predictive of cardiovascular disease and is significantly associated with morbidity and mortality. The mechanism by which angiotensin II (Ang II) and dietary sodium exert additive effects on the development of cardiac hypertrophy is unclear. The goal of this study was to evaluate the hypothesis that, where there is a genetic predisposition to Ang II-dependent hypertrophy, there is also an increased susceptibility to sodium-induced hypertrophy mediated by AT1-receptor expression.
Methods: Diets of low sodium (LS, 0.3% w:w) and high sodium (HS, 4.0% w:w) content were fed to adult (age 25 weeks) control wild-type mice (WT) and to transgenic mice exhibiting cardiac specific overexpression of angiotensinogen (TG). At the conclusion of a 40-day dietary treatment period, cardiac tissue weights were compared and the relative expression levels of Ang II receptor subtypes (AT1A and AT2) were evaluated using RT-PCR.
Results: WT and TG mice fed HS and LS diets maintained comparable weight gains during the treatment period. The normalised heart weights of TG mice were elevated compared to WT, and the extent of the increase was greater for mice maintained on the HS diet treatments (WT 12% vs. TG 41% increase in cardiac weight index). While a similar pattern of growth was observed for ventricular tissues, the atrial weight parameters demonstrated an additional significant effect of dietary sodium intake on tissue weight, independent of animal genetic type. No differences in the relative (GAPDH normalised) expression levels of AT1A- and AT2-receptor mRNA were observed between diet or animal genetic groups.
Conclusion: This study demonstrates that, where there is a pre-existing genetic condition of Ang II-dependent cardiac hypertrophy, the pro-growth effect of elevated dietary sodium intake is selectively augmented. In TG and WT mice, this effect was evident with a relatively short dietary treatment intervention (40 days). Evaluation of the levels of Ang II receptor mRNA further demonstrated that this differential growth response was not associated with an altered relative expression of either AT1A- or AT2-receptor subtypes. The cellular mechanistic bases for this specific Ang II-dietary sodium interaction remain to be elucidated.

JRAAS 2004;5:169-175.

POPULAR
TOPICPAPERAngiotensin II stimulates arachidonic acid release from bone marrow stromal cells
Renee S Richmond, E Ann Tallant, Patricia E Gallagher, Carlos M Ferrario, William B Strawn

Introduction: Angiotensin II (Ang II) is recognised as a regulator of haematopoiesis, but its actions within the bone marrow are not fully understood. Support of haematopoiesis by bone marrow stromal cells (MSC) is dependent on factors that include arachidonic acid and macrophage colony stimulating factor (MCSF), both of which are increased by Ang II stimulation in other tissues. To further elucidate the mechanisms of Ang II-regulated haematopoiesis, we determined whether Ang II-stimulation alters arachidonic acid release and MCSF secretion from MSC.
Methods: Cynomolgus monkey MSC isolated from bone marrow aspirates and the human HS-5 stromal cell line were studied for Ang II-mediated arachidonic acid (AA) release, while secretion of MCSF in response to Ang II was studied in HS-5 cells. Cells were labelled overnight with 3H-AA and the release of 3H-AA was measured in culture medium following 20 minutes stimulation with Ang II, alone or in combination with the AT1- or AT2-receptor antagonists, losartan and PD 123319, respectively. MCSF secretion into culture medium was measured using an enzyme immunoassay following 24 hours of treatment with Ang II alone or in combination with losartan or PD 123319. Phorbol-myristate-acetate, known to stimulate release of AA and MCSF, was used as a positive control in both experiments.
Results: In response to Ang II, release of 3H-AA from monkey and human MSC was increased (p<0.05) to 147±4% and 124±3% of control, respectively. The AT1- and AT2-receptor antagonists, losartan and PD 123319, individually reduced Ang II-stimulated 3H-AA release. In contrast, Ang II had no effect on secretion of MCSF from HS-5 cells.
Conclusions: These results provide mechanistic evidence for Ang II-mediated haematopoiesis through AA release that may, in part, explain Ang II-facilitated recovery of haematopoiesis in experimental myelosuppression and the anaemias associated with Ang II receptor blockade.

JRAAS 2004;5:176-182.

PAPEREffect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblasts
Paul Lijnen, Victor Petrov, Guillermo Diaz-Araya, Robert Fagard

Objective: The purpose of this investigation was to determine whether the aminopeptidase inhibitor with broad specificity, bestatin, affects angiotensin I (Ang I)-, angiotensin II (Ang II)- or angiotensin III (Ang III)-stimulated collagen gel contraction in cardiac fibroblasts.
Design and methods: Cardiac fibroblasts (from normal male adult rats) were cultured to confluency in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS). These fibroblasts (100,000 cells) were then further incubated in a floating collagen gel lattice with the test products Ang I (1 µmol/L), Ang II (100 nmol/L), Ang III (100 nmol/L) and bestatin (100 µmol/L) for three days in DMEM without FBS. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide.
Results: Ang I, II and III stimulated (p<0.05) collagen gel contraction by 30.4±4.8 (SEM)%, 27.1±3.1% and 15.4±3.6% respectively. Ang I- and II-induced stimulation of collagen gel contraction was of the same order but more pronounced (p<0.05) than Ang III- stimulated collagen gel contraction. The Ang I-, II- and III-stimulated collagen contraction was reduced by bestatin. Bestatin, however, did not affect basal collagen gel contraction in cardiac fibroblasts. Bestatin dose-dependently inhibited the hydrolysis of arginine- and alanine-p-nitroanilide in cardiac fibroblasts. When a neutralising antibody to transforming growth factor TGF-b1 was added to the collagen gel simultaneously with the angiotensins, the stimulated collagen contraction was not affected. Beta-aminoproprionitrile, an inhibitor of lysyl oxidase, completely abolished basal as well as Ang I-, II- and III-stimulated collagen contraction in cardiac fibroblasts.
Results: Our data suggest that aminopeptidases are involved in the Ang I-, II- and III-induced stimulation of collagen contraction in cardiac fibroblasts.

JRAAS 2004;5:183-188.

PAPERLectin chromatography of extrarenal renin protein in human plasma and tissues: Potential endocrine function via the renin receptor
Anthony JM Stubbs, Sandford L Skinner

Secretion of prorenin from extrarenal tissues comprises approximately half of the renin protein in plasma; its origin is unknown. The discovery of a prorenin/renin receptor that activates vascular tissue kinases raises interest in this otherwise inactive component. We have studied its glycosylation as this may distinguish it from renal renin. The binding of renin protein (active and prorenin) in human plasma and tissues to concanavalin (Con A) and wheat germ lectins was deployed. Immunoradiometric and enzyme kinetic assays were applied to column fractions. Mannosylated renin protein binds to Con A and has been shown to be taken up by human vascular and hepatic cells on mannose-6-phosphate receptors, possibly as a clearance mechanism. But the other binding sites of prorenin/renin that elicit a cellular phosphorylation response are apparently independent of glycosylation. The tissues examined (kidney, adrenal, ovary) each contain high proportions of Con A binding renin, but the plasma of normal resting males and females contain mainly non-binding renin, the proportion increasing as renal renin secretion decreases. The extreme of this relationship is seen in anephric patients and in some normal women on oral contraceptives with suppressed renal renin, in whom plasma renin is entirely non-binding to Con A. Conversely, when renal renin secretion was stimulated, the increased plasma active renin bound to Con A. However, extrarenal tissues containing exclusively Con A non-binding renin protein, and hence potential sources of anephric plasma renin protein, were not identified, but are unlikely to include adrenal or female reproductive tract. The findings are consistent with the view that normal human plasma contains a considerable amount of amannosylated prorenin of extrarenal origin that escapes hepatic clearance and has a longer half-life than renal renin. This plasma renin form would be expected to be activated in association with the recently described renin/prorenin aspartyl protease receptor and to participate in local pathophysiological processes.

JRAAS 2004;5:189-196.

PAPERRenal targeting of captopril using captopril-lysozyme conjugate enhances its antiproteinuric effect in adriamycin-induced nephrosis
Willemijn AKM Windt, Jai Prakash, Robbert Jan Kok, Frits Moolenaar, C Alex Kluppel, Dick de Zeeuw, Richard PE van Dokkum, Robert H Henning

Introduction: High-sodium intake blunts the renoprotective efficacy of angiotensin-converting enzyme (ACE) inhibitors. We investigated whether targeting the drug to the kidneys may attenuate the inferior response to ACE inhibitor (ACE-I) under high-sodium conditions. The ACE-I, captopril, was coupled to the low molecular weight protein (LMWP) lysozyme, yielding captopril-lysozyme conjugates that accumulate specifically in the proximal tubular cells of the kidneys.We compared the antiproteinuric efficacy of captopril to that of the captopril-lysozyme conjugate in adriamycin-induced proteinuric rats fed with a high-sodium diet.
Materials and methods: Rats with adriamycin (single injection 2 mg/kg)- induced proteinuria were put on a high-sodium diet (HS; 3% NaCl). When stable proteinuria developed at 5.5 weeks, animals were assigned to the following subcutaneous treatments: (1) vehicle (n=7); (2) lysozyme (equivalent to the amount in conjugate) (n=7); (3) captopril (5 mg/kg/24 hours) (n=8); (4) captopril-lysozyme conjugate (captopril content equivalent to 1mg captopril/kg/24 hours) (n=7). Blood pressure and proteinuria were monitored. After 10 days of treatment the rats were sacrificed and kidneys and plasma were removed.
Results: Results are given as mean + S.E.M. After injection with adriamycin at t=0, stable proteinuria developed, amounting to 547+79 mg/24 hours at week 5.5. Subsequently, after seven and nine days of treatment, no reduction of proteinuria was observed in the captopril-treated group. In contrast, a significant reduction in proteinuria, amounting to 35+4% (day seven) and 25+2% (day nine), was observed in the captopril-lysozyme conjugate group (p<0.05 compared with the captopril group). In contrast, blood pressure was reduced in the captopril-treated group by 13.9+2.9 mmHg, while in the captopril-lysozyme treated group, an increase of 7.9+3.3 mmHg was found. Renal ACE activity was lowered by 30% in the captopril, as well as in the captopril-lysozyme conjugate treated group, compared with control. Furthermore, the ratio of kidney: plasma levels of captopril almost doubled as a consequence of coupling to lysozyme.
Conclusion: In proteinuric rats fed with a high-sodium diet, captopril induced a reduction in blood pressure without an effect on proteinuria. In contrast, renal targeting of a five times lower dose of the ACE-I with the captopril-lysozyme conjugate reduced proteinuria without reducing blood pressure. Therefore, renal targeting of ACE-I may be a promising strategy to optimise the therapeutic response of ACE-I.

JRAAS 2004;5:197-202.

PAPERAngiotensin (1-7) re-establishes impulse conduction in cardiac muscle during ischaemia-reperfusion. The role of the sodium pump
Walmor C De Mello

Introduction: The effect of angiotensin (1-7) (Ang 1-7) on membrane potential and excitability of rat heart muscle under ischaemia/reperfusion was investigated.
Materials and methods: The hearts of adult rats were removed under deep anaesthesia and perfused using the Langendorff method. After 40 minutes of global no-flow ischaemia, the heart was reperfused for five minutes and the right ventricle was dissected out and transferred to a transparent chamber, through which normal oxygenated Krebs solution flowed continuously (37ºC). Measurements of membrane potential were performed using an intracellular microelectrode connected to a high impedance preamplifier. The muscle was stimulated with rectangular current pulses (3 ms duration; 0.6 Hz) generated by an electronic stimulator and isolation unit. To study the influence of Ang (1-7) on sodium pump current, isolated myocytes were voltage-clamped at -40 mV and the current generated by the pump was recorded before and after the administration of Ang (1-7) (10-8 M) to the bath.
Results: Ang (1-7) (10-8 M) hyperpolarised the ischaemic heart fibre and re-established impulse propagation. The increment of resting potential was related to the activation of the sodium pump. Indeed, Ang (1-7) (10-8 M) enhanced the transient outward current generated by an electrogenic sodium pump. Both effects of Ang (1-7) on membrane potential and pump current were abolished by ouabain (10-7 M). The cardiac refractoriness was also increased by Ang (1-7) (10-8 M).
Conclusions: Ang (1-7) activates the sodium pump, hyperpolarises the heart cell and re-establishes the impulse conduction during ischaemia/reperfusion. These effects of Ang (1-7), and the increment of cardiac refractoriness, provide an explanation for the reduced incidence of arrhythmias during ischaemia/reperfusion in the presence of Ang (1-7).

JRAAS 2004;5:203-208.

POPULAR
TOPICEDITORIAL REVIEWHow do thiazide and thiazide-like diuretics lower blood pressure?
Alun D Hughes

Thiazide diuretics are widely used for the treatment of hypertension, but the mechanism by which these drugs lower blood pressure in the long term remains unknown. This article reviews current knowledge about the hypotensive actions of thiazides and thiazide-like diuretics and discusses possible mechanisms of action.

JRAAS 2004;5:155-160.

POPULAR
TOPICEDITORIAL REVIEWThe revised role of ACE-inhibition after myocardial infarction in the thrombolytic/primary PCI era
Pieter J de Kam, Adriaan A Voors, Francesco Fici, Dirk J van Veldhuisen, Wiek H van Gilst

Many studies have investigated the process of left ventricular (LV) dilatation and the effects of angiotensin-converting enzyme (ACE) inhibitors after myocardial infarction (MI). It has been generally accepted that progression of LV dilatation is a major predictor of heart failure and death after MI. Also, attenuation of LV dilatation is thought to be one of the main mechanisms by which ACE inhibitors (ACE-Is) produce their beneficial effects. However, evidence for this hypothesis came from studies that were performed before thrombolytic therapy and primary percutaneous coronary intervention (PCI) were routinely used after acute MI. Nowadays, reperfusion is obtained much more frequently and LV dilatation after MI has become less prevalent. Nevertheless, ACE-Is proved effective in reducing cardiac morbidity and mortality. Therefore, mechanisms other than attenuation of LV dilatation, such as anti-atherosclerotic effects or plaque stabilisation, may explain the long-term beneficial effects of ACE-Is after MI.
In the present overview, we evaluate the role of LV dilatation and the effects of ACE-Is after MI in the thrombolytic/primary PCI era and provide recommendations on ACE-I use in clinical practice.

JRAAS 2004;5:161-168.