Canadian Journal of Physiology and Pharmacology

Dual Influence of Spontaneous Hypertension on Membrane Properties and ATP Production in Heart and Kidney Mitochondria in Rat: Effect of Captopril and Nifedipine, Adaptation and Dysadaptation


Hypertension is one of the so called "civilization diseases" affecting modern developed countries. Despite great efforts in prevention as well as in optimizing therapy, it still remains one of the most frequently present risk factors in cardiovascular diseases (Ryden et al. 2008). Thus, hypertension research remains the focus of many clinicians as well as pre-clinical investigators (Simko and Pechanova 2009). Concerning the heart in hypertension, much knowledge has accumulated with respect to the pathogenesis, and changes on the subcellular level (Kunes and Zicha 2006) as well as pharmacological therapy (Das and Harris 1992; Millgard et al. 1998; Meyers and Siu 2011). Unfortunately, however, little is known about hypertension-induced alterations in cellular bioenergetics in the myocardium (Waczulikova and Sikurova 2010). Moreover, the few available studies often present different views. For instance Postnov et al. (2007) emphasize the effects of disturbances in mitochondrial energy conversion, while Roman et al. (1992) emphasize adaptation to the disease. In addition, there is a lack of parallel studies dealing with hypertension-induced changes in bioenergetics in different organs. This is particularly so in the case of hypertension-induced changes in the properties and function of the mitochondria of the heart and kidney (Seppet et al. 2007), as kidneys play an important role in the pathophysiology of hypertension (De Cavanagh et al. 2003).

Several classes of drugs have been found effective for anti-hypertensive treatment, these include diuretics, angiotensin II receptor blockers (type AT1), inhibitors of angiotensin-converting enzyme (ACE), [beta]-receptor blockers, calcium entry blockers, etc. (Kyselovic 2005). They are also frequently used as combination therapy. In our present experiments we used either captopril (CAP) or a combination of captopril and nifedipine (CAP + NIF). In the selection of these drugs, we were influenced by their efficacy (Simko et al. 2009) as well as the frequency of use by researchers (Kyselovic 2005).

Clinical studies comparing mono versus combination therapies concentrate predominantly on the outcome in terms of blood pressure control, regression of cardiac hypertrophy, and mortality (Wald et al. 2009; Fox et al. 2011). Still, however, little is known about the influence of drug treatment on hypertension-induced changes in the function and properties of mitochondria. Consequently, additional information about spontaneouous development and the medication-induced changes in cellular and tissue bioenergetics of the hypertensive heart and kidney may be useful in the selection of an optimal therapy, particularly with regard to further diseases that may accompany hypertension.

In our study we started with the premise that persisting noxas, such as diabetes or hypertension, induce several endogenous protective responses in the tissues, which finally yield a certain degree of adaptation to the disease (Ravingerova et al. 2001; Ziegelhoffer et al. 1995).

To better understand the changes in tissue energetics that may be linked with adaptation to hypertension, we extended the investigation of mitochondrial ATP production in oxidative phosphorylation by estimation of the variables that may have a modulatory influence on mitochondrial function, such as fluidity, conjugated diene content, and the [Mg.sup.2+]-ATPase activity of mitochondrial membranes.

The goals of present study are to investigate (i) the effect of spontaneous hypertension on the properties, function, and ATP production of cardiac and kidney mitochondria; and (ii) the effect of treatment with captopril and the combined therapy with captopril and NIF on hypertension-induced alterations in the properties and function of the mitochondria of the heart and kidney.

Materials and methods

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (ILAR 1996), as well as the rules issued by the State Veterinary and Alimentary Administration of the Slovak Republic, based on paragraph 37 (6), legislation No. 488/2002 of the Slovak Parliament. The use of animals was also reviewed and approved by the animal care review committee at the Institute for Heart Research, Slovak Academy of Sciences, where the experiments were carried out.


Experiments were performed on 16-week-old male spontaneously hypertensive rats (SHR), purchased from AnLab Ltd., Prague, Czech Republic; a vendor recommended by the State Veterinary and Alimentary Administration of the Slovak Republic, and licensed for trade with animals in all European Communities. The strain of SHR was specified as Okamoto Kyoto - Charles River, with the code SH/NCRL. The code for Wistar rats used as normotensive controls was WKY/NCRL. Animals were treated for 4 weeks with the ACE inhibitor captopril (CAP, 80 mg x [(kg body mass).sup.-1] per oral (p.o.)) or with CAP plus the calcium entry blocking agent nifedipine (NIF, 10 mg x [(kg body mass).sup.-1] p.o). To keep the concentrations of the drugs on a more stable level, the daily doses were divided into 2 equal parts applied at 0700-0730 h and at 1900-1930 h. This was of particular importance in the case of NIF, the level of which decreases relatively quickly after administration. Untreated control animals obtained an equal volume of saline. Hypertensive animals were randomly allocated to one of the 3 groups: (i) SH/ NCRL rats treated with CAP alone (n = 45), (ii) SH/NCRL treated with CAP + NIF (n = 45), or (iii) untreated control SHR (n = 96). Simultaneous testing was performed with Wistar rats (n = 96) as the normotensive controls. Both the normotensive and the hypertensive control groups were further divided among 2 groups (n = 48 animals); 12 rats from each of these groups were investigated at 16 weeks old, the remaining 12 were investigated at 20 weeks.

All animals were kept under a standard 12 h (light)--12 h (dark) regimen at 22 [+ or -] 2 [degrees]C. They were fed with a standard pellet diet, ad libitum, and had unrestricted access to drinking water. Systolic blood pressure (SBP) and heart rate of rats were monitored using the tail cuff technique (AD Instruments PowerLab[R], Germany) at the beginning, on every 7th day, and before termination of the experiment.

Because of considerable seasonal differences in the functional properties and some enzyme activities of cardiac mitochondria (Mujkosova et al. 2008), all experiments with isolated mitochondria were performed in blocks not exceeding 4-6 weeks, in the same season, starting in October and ending at the beginning of May.

Removal of hearts and kidneys

Animals were weighed, treated with heparin (Zentiva, Leciva, 100 mg x [(kg body mass).sup.-1], by intraperitoneal injection (i.p.)) and were anesthetized with thiopental (Valeant Czech Pharma; 45 mg x [kg.sup.-1]). After bilateral thoracotomy and opening of the abdomen, the hearts and kidneys were quickly excised. Hearts were cooled down and washed free of blood with an ice-cold solution containing 180 mmol x [L.sup.-1] KCl, 4 mmol x [L.sup.-1] EDTA, and 10 mmol x [L.sup.-1] Tris, pH 7.4. Isolation medium used for kidneys, contained 0.23 mol x [L.sup.-1] mannitol, 0.07 mol x [L.sup.-1] sucrose, 1 mmol x [L.sup.-1] EDTA, and 10 mmol x [L.sup.-1] Tris-HCl, at pH 7.4 (as recommended in de Cavanagh et al. 2003). After removal of the teguments, blood vessels, and fat, hearts were first weighed for estimation of the heart to body mass ratio (HW/BW) and then mitchondria were immediately isolated from both the hearts and kidneys.

Isolation of mitochondria from the heart

Hearts were moistened with a small volume of ice-cold isolation solution containing 180 mmol x [L.sup.-1] KCl, 4 mmol x [L.sup.-1] EDTA, and 1% bovine serum albumin, at pH 7.4, and subsequently cut into small pieces with scissors. The minced tissue was then transferred to a beaker with 20 mL of the isolation solution containing an additional 2.5 mg x [(g heart wet weight).sup.-1] of protease (Sigma P-6141). After 20 min of protease treatment with mild stirring, the whole suspension was transferred to a teflon-glass homogenizer and homogenized gently for 2-3 min. After centrifugation at 1000g for 10 min, the protease-containing supernatant was discarded together with the mitochondria that were in direct contact with the protease. This procedure was repeated once more. The pellet was resuspended in the same volume of the protease-free isolation solution. This suspension, now mostly containing mitochondria that were not in direct contact with protease, was spun down at 5000g for 15 min. The pellet containing mitochondria was again resuspended in an albumin-free isolation solution containing 180 mmol x [L.sup.-1] KCl and 4 mmol x [L.sup.-1] EDTA. It was spun down at 5000g for 15 min and subsequently used for estimation of protein concentration, according to the methods of Lowry et al. (1951), as well as for further biochemical and biophysical estimations. The whole isolation procedure was performed at 4 [degrees]C. With the exception of the protease treatment, the latter isolation procedure was essentially similar to that applied in our earlier study (Ziegelhoffer et al. 2002).

Isolation of mitochondria from the kidneys

Clean kidney tissue was minced as for the heart tissue, then it was transferred directly to a teflon-glass homogenizer together with 20 mL of isolation medium, and homogenized gently for 2-3 min. …

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