From the Division of Hypertensive and Vascular Medicine, University Hospital of Lausanne, Switzerland.
Abstract
We assessed the blockade of the renin-angiotensin system (RAS) achieved with 2 angiotensin (Ang) antagonists given either alone at different doses or with an ACE inhibitor. First, 20 normotensive subjects were randomly assigned to 100 mg OD losartan (LOS) or 80 mg OD telmisartan (TEL) for 1 week; during another week, the same doses of LOS and TEL were combined with 20 mg OD lisinopril. Then, 10 subjects were randomly assigned to 200 mg OD LOS and 160 mg OD TEL for 1 week and 100 mg BID LOS and 80 mg BID TEL during the second week. Blockade of the RAS was evaluated with the inhibition of the pressor effect of exogenous Ang I, an ex vivo receptor assay, and the changes in plasma Ang II. Trough blood pressure response to Ang I was blocked by 35±16% (mean±SD) with 100 mg OD LOS and by 36±13% with 80 mg OD TEL. When combined with lisinopril, blockade was 76±7% with LOS and 79±9% with TEL. With 200 mg OD LOS, trough blockade was 54±14%, but with 100 mg BID it increased to 77±8% (P<0.01). Telmisartan (160 mg OD and 80 mg BID) produced a comparable effect. Thus, at their maximal recommended doses, neither LOS nor TEL blocks the RAS for 24 hours; hence, the addition of an ACE inhibitor provides an additional blockade. A 24-hour blockade can be achieved with an angiotensin antagonist alone, provided higher doses or a BID regimen is used.
Key Words: angiotensin II • receptors, angiotensin • angiotensin-converting enzyme • human • telmisartan • losartan
Introduction
Blockade of the renin-angiotensin system with angiotensin (Ang) II AT1 receptor antagonists is now recognized as an effective means of lowering blood pressure in hypertensive patients.1 In addition, several large clinical trials performed with these agents have demonstrated that blocking AT1 receptors can confer a benefit in terms of morbidity and/or mortality in patients with essential hypertension and left ventricular hypertrophy2 as well as in patients with type 2 diabetic nephropathy3–5 and congestive heart failure.6,7
Defining the adequate dose of an antihypertensive agent has always been a difficult task.8 This difficulty has to do mostly with methodological problems such as blood pressure measurements or trial designs. Thus, for example, relatively low doses of ACE inhibitors are often sufficient to lower blood pressure, and it is not always evident that increasing the dose of these agents provides significantly more antihypertensive efficacy.9 Closely linked with the search of the optimal dose of an ACE inhibitor is the question of whether angiotensin conversion in plasma and tissues can be blocked completely and what dose is needed to achieve this goal. With time, it has become evident that Ang II virtually disappears from the circulation at peak of initial ACE inhibition but that Ang II levels in plasma remain measurable with long-term therapy and are often close to pretherapeutic levels at trough.10–12 These observations suggest that it is difficult to obtain a complete and persistent blockade of the renin-angiotensin system with ACE inhibitors.
Today, a similar debate is developing with Ang II receptor antagonists. Although all angiotensin antagonists have been shown to inhibit the effect of exogenous Ang I or II dose dependently at peak, the dose recommendations for the clinical use have been based on their antihypertensive efficacy, and, as for many other antihypertensive drugs, angiotensin antagonists are considered to have a rather "flat" dose-response curve.1 Yet, studies have demonstrated that the recommended doses of several Ang II receptor antagonists do not provide a full blockade of AT1 receptors around the clock and not even at peak.13,14 Thus, several investigators have proposed to associate an Ang II receptor antagonist with an ACE inhibitor to block the renin-angiotensin cascade at 2 sites and hence to obtain a greater and longer-lasting blockade of the system.15–17 However, no study has evaluated whether doses of Ang II receptor antagonists beyond those recommended for the treatment of hypertension can produce as much blockade of the renin-angiotensin system as an association of angiotensin receptor antagonists and ACE inhibitors.
To answer this question, we compared the blockade of the renin-angiotensin system produced by 2 Ang II receptor antagonists, losartan and telmisartan, administered either alone according to different dose regimens or in association with an ACE inhibitor in normotensive healthy volunteers.
Methods
Subjects
Thirty healthy male subjects took part in 2 consecutive studies. In the first study, 20 normotensive volunteers 26.5 years of age (range, 19 to 35), with a body mass index of 22.2±1.7 kg/m2 (mean±SD; range, 19.1 to 25.3) were enrolled. The second protocol involved 10 volunteers 26.5 years of age (range, 20 to 33), with a body mass index of 21.7±2.8 kg/m2 (range, 18.6 to 26.6).
All subjects were considered healthy on the basis of medical history, physical examination, routine blood and urine analyses, and an ECG. The study protocols were approved by our institutional review committee. Written consent was obtained from each volunteer after explanation of the nature, purpose, and potential risks of the study.
Study Design
Protocol 1
In this single-blind study, 20 volunteers were randomly assigned to 2 parallel groups of 10 subjects (). During the first week, they received either losartan (100 mg OD) or telmisartan (80 mg OD). The second week, lisinopril (20 mg OD) was added to the first week’s regimen. Blockade of the renin-angiotensin system was assessed at 4 and 24 hours on day 0 without drug intake and again at 4 and 24 hours after the last drug intake at the end of the first and second week of treatment. Subjects continued their usual free sodium intake throughout the study, but consumption of caffeine-containing beverages, alcohol, or smoking was not allowed the day before and during the investigational days.
fig-ommitted
Figure 1. Design of protocols 1 and 2.
On each investigational day, the volunteers were asked to come to our research facility after an overnight fast. They were comfortably installed in a supine position, and a venous catheter was placed in each forearm, one for blood sampling and the other for Ang I injections. At each time point (4 and 24 hours), the blood pressure response to exogenous Ang I was tested as described previously.13 In addition, blood was taken to measure plasma Ang II levels and the blockade of AT1 receptors ex vivo by using an in vitro Ang II receptor-binding assay on times 0, 4, and 24 hours. During the entire study, the volunteers were asked to return every morning to receive the drug under supervision.
Protocol 2
In this single-blind study, 10 volunteers were randomly assigned to 2 parallel groups of 5 subjects (). During the first week they received either losartan (200 mg OD) or telmisartan (160 mg OD). The second week, the same dose of each drug was given, but according to a twice-a-day regimen (losartan, 100 mg BID, and telmisartan, 80 mg BID). The same assessment of Ang II receptor blockade was performed.
Blood Pressure Measurement, In Vitro Assessment of Ang II Receptor Blockade, and Plasma Ang II
Blood pressure and heart rate were monitored noninvasively by photoplethysmography at the finger (Finapres, Ohmeda), as described previously.18 To measure plasma Ang II levels, an immunoreactive method with monoclonal antibodies against Ang II was used.19 Ex vivo Ang II receptor blockade assessment was performed with the use of a standardized in vitro receptor assay, as described previously.20
Statistical Analysis
All results are mean±SD unless otherwise specified. One-way ANOVA was performed followed by either paired or unpaired t tests with the use of GraphPad Prism, version 3.00, for Windows. A probability value <0.05 was considered to indicate statistical significance.
Results
All subjects completed the study. The drugs were well tolerated, and no clinically significant adverse effect or change in safety parameters (hematological, hepatic, renal, or ECG) was recorded.
Protocol 1
Changes in trough systolic and diastolic blood pressure with the various treatments in the 2 groups are shown in . When given alone, 100 mg losartan once daily did not produce any significant change in blood pressure, whereas telmisartan alone induced a significant decrease in systolic blood pressure, but baseline blood pressure was higher in the telmisartan group. With the addition of the ACE inhibitor, significant changes in blood pressure were observed both with losartan and telmisartan.
fig-ommitted
TABLE 1. Protocol 1: Changes in Trough Blood Pressure After One Week of Treatment
The effects of the different treatment regimens on the blockade of the renin-angiotensin system as assessed by the 3 methods are shown in . Four hours after the last drug intake, the mean blood pressure response to Ang I was significantly blocked with losartan and telmisartan. However, at 4 hours, the blockade induced by 100 mg OD losartan was greater than that induced by 80 mg OD telmisartan (P<0.01). At trough, blockade was significantly lower with both agents, and no significant difference was found between the 2 compounds. With the adjunction of 20 mg OD lisinopril, the antagonism was higher and similar in both groups at 4 hours (P<0.05 versus losartan alone and P<0.01 versus telmisartan alone) as well as at 24 hours (P<0.01 versus either drugs alone). With the association, the 4-hour blockade was almost complete, and, most importantly, the trough effect remained substantial, as it was equivalent to the peak effect of 100 mg OD losartan.
fig-ommitted
TABLE 2. Blockade of the Renin-Angiotensin System as Assessed by 3 Different Methods in Protocol 1
The ex vivo receptor binding assay results supported the in vivo data but differed in that the trough AT1 receptor blockade results were lower in the losartan group. Because lisinopril does not interact with the AT1 receptor, this assay enables us to specifically evaluate the degree of blockade attributable to the angiotensin receptor antagonist alone. Thus, as expected, no difference was observed with or without the addition of the ACE inhibitor. The reactive rise in plasma Ang II levels also supported our in vivo results. Four and 24 hours after the last drug intake, significant increases in plasma Ang II versus baseline were obtained with both 100 mg OD losartan (P<0.05 and P<0.01, respectively) and 80 mg OD telmisartan (P<0.01 at both time points). There was no statistically significant difference when both drugs were compared. With the addition of lisinopril, Ang II levels returned to the baseline values at 4 hours in both groups, reflecting the blockade of ACE. At 24 hours, the effect of lisinopril had decreased, and, consequently, Ang II levels were significantly higher than baseline levels though markedly lower than with the Ang II antagonists alone.
Protocol 2
The changes in systolic and diastolic blood pressures are shown in . The fall in blood pressure was particularly marked when volunteers received 100 mg BID losartan (-11±4 mm Hg systolic blood pressure and -10±3 mm Hg diastolic blood pressure, P<0.01 versus baseline), but the number of subjects is too small to demonstrate a significant difference with other treatment regimens, and baseline blood pressure was in this case higher in the losartan group.
fig-ommitted
TABLE 3. Protocol 2: Changes in Trough Blood Pressure After One Week of Treatment
The degree of blockade of the renin-angiotensin system in protocol 2 are presented in . At 4 hours, the blood pressure response to exogenous Ang I was blocked by 90% with 200 mg OD losartan and 72% with 160 mg OD telmisartan. The blockade induced by telmisartan was significantly lower than that induced by losartan (P<0.01). At trough, Ang I receptor blockade was similar with both compounds when these were given once per day. The twice-a-day schedule for losartan (100 mg BID) provided a degree of blockade at 4 hours similar to the once-a-day administration (200 mg OD), but the trough blockade was significantly greater with the BID regimen (P<0.01). With telmisartan, 160 mg OD was equivalent to 80 mg BID.
fig-ommitted
TABLE 4. Blockade of Renin-Angiotensin System Assessed by 3 Different Methods in Protocol 2
When assessed with the ex vivo test, Ang II receptor blockade was again more sustained with the BID regimen of losartan, whereas no difference between OD and BID was found with telmisartan. The data also suggest that at 4-hour receptor blockade with telmisartan 80 or 160 mg is not complete. The changes in plasma Ang II levels were also consistent with our in vivo results in losartan group.
Discussion
Several recent studies have suggested that combining an ACE inhibitor with an Ang II receptor blocker is more effective to block the renin-angiotensin system than either substance given alone.15–17,21,22 However, none of these studies have used doses of Ang II receptor antagonists higher than those recommended for the treatment of essential hypertension. We have therefore designed our study to assess whether higher doses of an Ang II receptor blocker could be as effective as the association of a usual dose of the antagonist combined with an ACE inhibitor in blocking the vasopressor effects of Ang II. Because the main goal of the study was to investigate the blockade of the renin-angiotensin system (including with injections of exogenous Ang I) rather than the changes in blood pressure, the study was conducted in normotensive subjects. As summarized in , our results clearly demonstrate that a complete 24-hour blockade of the Ang II effects cannot be achieved with the recommended doses of losartan (100 mg) and telmisartan (80 mg) given once per day. Hence, with these dosing regimens of antagonists, the combination with an ACE inhibitor not surprisingly has an additive effect to provide an almost complete and long-lasting blockade of the renin-angiotensin system. However, a comparable sustained 24-hour inhibition of the system can be obtained with losartan alone when 100 mg of the drug is given twice per day. Despite its longer duration of action, 160 mg OD or 80 mg BID telmisartan did not provide a complete blockade of the system throughout the day.
fig-ommitted
Figure 2. Summary of the blockade of the blood pressure (BP) response to exogenous Ang I at trough in the different study groups (n=5 to 10 according to protocols). Data are mean±SD; *P<0.01.
ACE inhibitors as well as Ang II receptor antagonists have been developed to block the multiple effects of Ang II possibly throughout the day and hence to lower blood pressure. With time, however, it became evident that continuous 24-hour blood pressure control could be achieved with ACE inhibitors despite intermittent resumption of normal ACE activity.23 Furthermore, during chronic ACE inhibition and particularly at trough, circulating Ang II levels are not at all suppressed, thus emphasizing the difficulty to block the renin-angiotensin system completely over a long period of time.10–12 Such an apparent discrepancy between the duration of blockade and of the antihypertensive effect appears to exist also with the use of Ang II receptor antagonists. Indeed, almost all Ang II receptor blockers including losartan and telmisartan have been shown to lower blood pressure over 24 hours when given once per day in patients with mild to moderate hypertension.1 Yet, most antagonists do not block the renin-angiotensin system around the clock.13,14 In this study, we have investigated 2 angiotensin receptor antagonists, one with a very long duration of action, telmisartan, and another with a shorter duration of action, losartan. Our data demonstrate that neither telmisartan nor losartan are capable to produce a complete blockade of the renin-angiotensin system for 24 hours when used at their maximal recommended doses of 80 and 100 mg, respectively. The results obtained with 100 mg OD losartan in this study are comparable to those reported previously that used the same methodology with a 70% inhibition at 4 hours and a 35% residual blockade at trough.24 The 36% blockade of the blood pressure response to exogenous Ang I at trough with 80 mg telmisartan was surprising. However, our results are in agreement with the recent results of Stangier et al,25 who performed a complete telmisartan dose-response curve in normotensive subjects. In this latter study, the reactive rise in plasma Ang II levels was also comparable to our results if one takes into account the methodological differences.
The blockade of the renin-angiotensin system being only partial with 100 mg losartan and 80 mg telmisartan, it is not surprising that the association with 20 mg lisinopril is additive and produces a complete blockade at 4 hours and a 75% inhibition at trough with both antagonists. These findings are therefore in agreement with previous observations suggesting that the combination is more effective in antagonizing the system than a single site inhibition in normotensive subjects.15,16 At this point, it is important to mention that the intrinsic variability of blood pressure is such that the blood pressure response to exogenous angiotensin can hardly be 100%. Indeed, even during complete blockade, there are small physiological fluctuations of blood pressure. In our hands, this spontaneous variability of blood pressure in normotensive subjects averages {approx} 13% (ie, 3 to 4 mm Hg); hence, a blockade >85% can be considered as complete.
Previous studies with ACE inhibitors and Ang II receptor antagonists have shown that increasing the dose once daily has little effect on the peak inhibition but tends to prolong the duration of the inhibition.14,18,26 In accordance with this observation, increasing the dose of losartan to 200 mg OD and that of telmisartan to 160 mg OD significantly improved the trough blockade, with only a slight improvement in the degree of blockade measured 4 hours after drug intake. Yet, even though both drugs were administered for 1 week, the trough blockade remained far from being complete, whatever the method of assessment.
The main goal of these experiments was to demonstrate that an Ang II receptor antagonist alone can be as effective as an ACE inhibitor–Ang II receptor blocker association, provided that it is administered at the right dose and dosing interval. Our results show that this is indeed the case. When 100 mg losartan was given twice per day, the degree of antagonism was similar to that of the combination of 100 mg losartan with 20 mg lisinopril. Interestingly, this was not the case with 80 mg BID telmisartan. The difference is possibly explained by the very long half-life of telmisartan.27 With long-acting drugs, a twice-a-day regimen does not provide any benefit, and it would certainly be more adequate to increase the dose further. It is conceivable that higher doses of telmisartan (>160 mg/d) once daily would enable the same degree of antagonism to be reached as with 100 mg BID losartan.
Taken together, our results demonstrate that an equal 24-hour blockade of the renin-angiotensin cascade can be obtained with an Ang II receptor blocker alone as with an ACE inhibitor/Ang II receptor blocker combination. However, to achieve this goal with a monotherapy, it implies the use of higher doses of the antagonists and in some cases a twice-a-day regimen. One may also be concerned by safety considerations when administering higher doses of AT1 receptor blockers. This is hardly an issue, since AT1 receptor antagonists do not exhibit any dose-dependent adverse effects. Indeed, AT1 receptor blockers have been used safely at higher doses. For example, in the ValHeft trial, many patients received 160 mg valsartan twice a day without any safety concerns,7 and the high doses used in this trial probably explain the marked efficacy observed in the ACE-intolerant patients. On the other hand, the use of angiotensin receptor antagonists alone rather than in association with ACE inhibitors avoids all the side effects associated with this latter class of drugs. This study has some limitations. First, it was conducted in young normotensive subjects who have a reactive renin-angiotensin system. One may thus argue that our observations do not necessarily apply to patients with a less active renin-angiotensin system, such as older hypertensive patients or patients receiving concurrent ß-blockade. Second, subjects have been studied on a free sodium intake and the degree of blockade of the renin-angiotensin system by AT1 receptor antagonists may well vary, depending on the baseline activation of the system. Last, inhibition of the renin-angiotensin system hardly causes a decrease in blood pressure in normotensive subjects. Hence, it is difficult to demonstrate that a greater inhibition provides a greater antihypertensive efficacy. Yet, in our subjects, a significant correlation was found between the percent inhibition at trough and the percent change in systolic blood pressure (r=-0.25, P=0.05, n=60).
Perspectives
Although they were obtained in normotensive subjects, our observations may have some clinical implications, as they may help defining the most adequate dose of Ang II receptor blocker to use clinically. Indeed, today, the recommended doses of ACE inhibitors and Ang II receptor blocker have been chosen on the basis of their ability to lower blood pressure. However, the recent trials investigating the ability of these agents to protect patients against target organ damage have now repeatedly shown that the highest doses were most effective,2–5,7,28 thus recommending more aggressive treatment in the future. These studies have also suggested that there may be benefits of blocking the effects of Ang II beyond blood pressure control.2 This would indicate that the doses used for blood pressure control are not those needed for target organ protection. If this hypothesis is true, a complete 24-h blockade of the renin-angiotensin system would definitely be a better target for treatment, and the results obtained with one antagonist may not necessarily be assumed to be obvious for all others. However, whether a full 24-hour blockade of the renin-angiotensin system provides a better organ protection than a transient blockade remains to be further investigated prospectively in clinical trials.
Acknowledgments
This work was supported by a grant from Boehringer-Ingelheim (Switzerland). The authors thank Monique Salvi and Françoise Nicoud for excellent assistance.
Received August 12, 2002; first decision September 7, 2002; accepted November 6, 2002.
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PKC in Cell Membranes of Elderly HypertensivesPablo V. Escribá; José M. Sánchez-Dominguez; Regina Alemany; Javier S. Perona; Valentina Ruiz-Gutiérrez
From the Laboratory of Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands (P.V.E., R.A.), Palma de Mallorca, Spain; and Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (J.M.S.-D., J.S.P., V.R.-G.), Sevilla, Spain.
Correspondence to Pablo V. Escribá, Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. Valldemossa Km 7.5, E-07071 Palma de Mallorca, Spain.
Abstract
In this study, we quantified the levels of lipids and signaling proteins in erythrocyte membranes from elderly normotensive and hypertensive subjects. In hypertensive subjects, the cholesterol/phospholipid ratio increased significantly in erythrocyte membranes, owing to the reduction of phospholipid levels concomitant with a rise in the levels of cholesterol. In addition, differences were also found in the amount of fatty acids in both phospholipid and cholesterol esters. Erythrocyte membranes from hypertensive subjects contained higher levels of monounsaturated and lower levels of polyunsaturated fatty acids. On the other hand, signaling proteins such as G proteins and protein kinase C have been implicated in the control of blood pressure. Previous studies have shown that the cellular localization and the activity of these proteins are modulated by the type and the abundance of membrane lipids. For this reason, we assessed the levels of these signaling molecules in the membrane. We found that the levels of membrane-associated (active/preactive) G proteins (G{alpha}
i, G{alpha}
o, and Gß) and protein kinase C were significantly reduced in hypertensive subjects. We believe that these alterations could be related to the etiopathology of hypertension in elderly subjects or alternatively may correspond to adaptive compensatory mechanisms.
Key Words: blood pressure • G proteins • elderly • protein kinases • membranes • lipids
Introduction
Cardiovascular pathologies constitute the main cause of death in industrialized countries.1 Among the different manifestations and symptoms that arise during the development of cardiovascular illnesses, hypertension is a major risk factor,2 the control of which is one of the main aims of cardiovascular therapies.3 Multiple metabolic abnormalities accompany essential hypertension, and these include alterations in serum lipoprotein levels (HDL, LDL and VLDL), hypertriglyceridemia, hypercholesterolemia, insulin resistance, and so forth.4–6 In addition, alterations in the composition and properties of membranes have been reported both in hypertensive humans7–11 and animal models of hypertension.12–14 Modifications of membrane lipids change the physical and functional properties of the cell barrier, and such modifications could be reflected in alterations of cellular physiology in hypertensive subjects. Indeed, the cellular localization and activity of G proteins and protein kinase C (PKC) are modulated by membrane lipids and drugs that modulate the membrane structure.15,16
G proteins and PKC are signaling elements critical in the propagation of messages from G-protein–coupled receptors (GPCRs). The relevance of G proteins and PKC in the control of blood pressure is reflected in the fact that a wide variety of GPCRs are involved in this physiological process. In this context, {alpha}
1-, {alpha}
2-, and ß-adrenoceptors have been shown to control blood pressure at different levels. The {alpha}
2-adrenoceptor controls blood pressure centrally by activating Gi proteins, whose {alpha}
-subunit inhibits the production of cAMP by adenylyl cyclase.17,18 In contrast, {alpha}
1- and ß-adrenoceptors control blood pressure peripherally, activating phospholipase C and adenylyl cyclase through G{alpha}
o/q and G{alpha}
s proteins, respectively. The effector proteins phospholipase C and adenylyl cyclase regulate intracellular levels of second messengers, such as diacylglycerol, inositol phosphate, and cAMP. These second messengers, along with Gß{gamma}
-protein subunits, modulate the activity of other messengers, including various protein kinases (PKC, PKA, and GRK) that phosphorylate and modulate the activity of several proteins. Some of the targets of these kinases are activated GPCRs that, on phosphorylation, become desensitized.19–21
In this scenario, alterations in membrane lipids and of signaling membrane proteins could be relevant to the pathophysiology of hypertension.22,23 On one hand, hypertension has been associated with multiple alterations in the physical properties of the plasma membrane.24 On the other hand, an impairment of the adrenoceptor/G protein/adenylyl cyclase system has been consistently observed in hypertensive humans and animals, although the molecular basis of these alterations remains largely unknown.25–29
This study was designed to evaluate the status of serum and membrane lipids as well as membrane-associated signaling proteins (G proteins and PKC) in erythrocytes of elderly normotensive and hypertensive subjects. Between these two groups, we found significant differences in the levels of membrane lipids, G proteins, and PKC. In erythrocytes from hypertensive subjects, an increase in the levels of membrane cholesterol and a decrease in total phospholipids was found, as were alterations in the relative abundance of their fatty acid moieties. Immunochemical quantification of membrane-bound G proteins revealed decreases in different G-protein subunits. Finally, the amount of PKC{alpha}
was significantly lower in hypertensive subjects with respect to normotensive subjects.
In summary, this work presents relevant data that could explain the alterations of GPCR signaling associated with hypertension in elderly (and possibly other) subjects. It is likely that alterations in membrane lipid levels in hypertensive subjects affected the localization and activity of G proteins, PKC, and possibly other signaling proteins. These changes could explain the observed impairment in signal propagation from GPCRs in hypertensive subjects.
Methods
Subjects
Elderly hypertensive and normotensive (control) subjects were studied. The hypertensive and control groups each consisted of 28 subjects (). Blood pressure was measured with a mercury-gauge sphygmomanometer on the right brachial artery of each subject. At each visit, 3 blood pressure readings were recorded, and the average was used to determine the eligibility of the subject. In addition, the subject’s blood pressure was recorded at the beginning and end of each period of study. The criterion for hypertension was a systolic blood pressure ">="
140 mm Hg and a diastolic value of ">="
90 mm Hg on at least 3 different occasions. Blood pressure was recorded after the subject had been at rest in a supine position for 10 minutes.30 Before recruitment, the medical histories of all the participants were reviewed comprehensively, and a physical examination and clinical chemical analysis were performed to exclude possible secondary causes of hypertension. None of the subjects used in the study had diabetes mellitus or hypothyroidism, and no history of alcohol abuse or cigarette smoking was seen. All subjects gave their informed consent before participating in the study. Hypertensive subjects were treated with enalapril, felodipine, spironolactone, or amiloride, but none of these drugs has been reported to alter the levels of the proteins studied here. Biochemical and physiological parameters indicated that the control group was healthy. All the protocols used in this study were approved by the Institutional Committee of Human Research (Comité de Ensayos Clínicos, Hospital Universitario Virgen del Rocío, Sevilla, Spain).
fig-ommitted
TABLE 1. Characteristics of Hypertensive and Control Subjects
Biochemical and Plasma Lipid Measurements
Levels of blood glucose, creatinine, and uric acid were measured by conventional enzymatic methods, with the use of venous blood obtained on the day of the examination after overnight fasting. Similarly, LDL, HDL, and total cholesterol, phospholipids, and triglycerides in serum were measured by enzymatic methods.31,32
Preparation of Erythrocytes
Erythrocyte membranes were prepared as described previously.4–8 Briefly, blood samples obtained after overnight fasting were collected in heparinized tubes and centrifuged at 1750g at 4°C for 10 minutes. The plasma and buffy coat were removed, and the erythrocyte pellet was washed twice with 110 mmol/L MgCl2. Aliquots of this preparation were used to simultaneously determine the membrane lipid and protein content.
Analysis of Lipid Classes and Fatty Acid Methyl Esters
Quantitative extraction of total erythrocyte membrane lipids from 5 mL of blood was carried out as described elsewhere.33–35 Lipid extracts were dissolved in chloroform/methanol (2:1 vol:vol) and passed through 0.2-µm filters. They were then analyzed by liquid chromatography, as described previously, with the use of standard solutions for both identification and quantification.4–11 Lipids were also separated by thin-layer chromatography on silica gel plates (Kieselgel 60 F254, Merck). The mobile phase was a mixture of hexane/diethylether/acetic acid (80:20:1 vol/vol/vol), as described.36 The phospholipid and cholesteryl-ester fractions were transmethylated, and the resulting fatty acid methyl esters were analyzed by gas chromatography.36 Individual fatty acid methyl esters were identified by comparison with known standards or by gas chromatography–mass spectrometry.
Immunoblot Analysis and Quantification of G Proteins and PKC{alpha}
Immunoblotting of G proteins and PKC{alpha}
from erythrocyte membranes of elderly normotensive (control) or hypertensive subjects was performed as described elsewhere.37 Briefly, a pellet of erythrocytes from 1 mL of blood was combined with 1 mL of homogenization buffer (50 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA, 2 mmol/L MgCl2, 1 mmol/L PMSF, and 5 mmol/L iodoacetamide) and homogenized with a blade-type homogenizer. The samples were then centrifuged at 600g and 4°C for 5 minutes. The pellets that contained whole cells were discarded, and the supernatants were then centrifuged twice at 40 000g and 4°C for 20 minutes. These final pellets contained the erythrocyte membranes, and they were resuspended in 500 µL of homogenization buffer and 250 µL of solubilization buffer (160 mmol/L Tris-HCl [pH 6.8], 8% sodium dodecyl sulfate). The protein content of these samples was then determined by means of the bicinchoninic acid method.38 Finally, the sample proteins were separated by electrophoresis and immunoblotted with the following specific primary antibodies, as described elsewhere37: anti-G{alpha}
i1/2 (1:7000 dilution), anti-G{alpha}
s (1:5000; used to measure the 52-kDa band),37,39 anti-G{alpha}
o (1:4000), and anti-Gß (1:5000) were from New England Nuclear Corp, and anti-PKC{alpha}
(1:1000) was from BD Transduction Laboratories. The primary antibodies were detected with horseradish peroxidase–linked secondary antibodies (1:2000 dilution; Amersham Pharmacia) and visualized with the enhanced chemiluminescence Western blot detection system (Amersham Pharmacia) exposed to enhanced chemiluminescence hyperfilm (Amersham Pharmacia). Quantification was performed by image analysis as described.37
Statistical Analysis
The results of the statistical analyses are expressed as mean±SEM. One-way ANOVA, followed by the Scheffé test, was used for statistical evaluations. Differences between experimental groups were considered statistically significant at a value of P<0.05.
"" hspace=5
Results
The demographic characteristics, blood pressure, and serum lipid/lipoprotein levels of the subjects used in this study are shown in . The mean±SEM levels of glucose, creatinine, and uric acid in control subjects were 101±3 mg/dL, 0.97±0.18 mg/dL, and 4.9±1.4 mg/dL, respectively. In hypertensive subjects (n=28), these values were 95±13 mg/dL, 1.1±0.24 mg/dL, and 5.5±1.5 mg/dL, respectively. In the control group, mean±SEM blood pressure was 140±4.0 mm Hg (n=28), a normal value for subjects of this age (86±1 years). The hypertensive group had a significantly higher systolic blood pressure value (145.2±4.2 mm Hg, P<0.001, n=28) (). Diastolic blood pressure was also significantly higher in elderly hypertensive subjects (72.4±2.1 mm Hg and 76.6±2.2 mm Hg for normotensives and hypertensives, respectively; P<0.001) ().
Serum Lipoproteins in Elderly Hypertensive Subjects
Although no significant differences in total cholesterol were observed, a significant decrease in the levels of HDL-cholesterol was found in elderly hypertensives (56.8±3.5 mg/dL and 51.7±3.2 mg/dL in normotensive and hypertensive subjects, respectively, P<0.001) (). Increased levels of serum triglycerides were also found in this group (77.8±5.3 mg/dL and 94.6±6.4 mg/dL for normotensive and hypertensive subjects, respectively; P<0.01). In contrast, the hypertensive group had lower levels of phospholipids (182±5 mg/dL and 178±7 mg/dL in normotensives and hypertensives, respectively, P<0.05) (). These alterations are characteristic features of hypertensive subjects, further evidence of their pathological status.4–8
Erythrocyte Membrane Lipids in Elderly Hypertensive Subjects
Erythrocyte membranes from elderly hypertensive subjects contained a higher percentage of cholesterol with respect to the total lipid content in normotensive and hypertensive subjects, respectively (23.5±1.5% and 25.8±0.9%; P<0.05; ). In contrast, the percentage of phospholipids was lower in hypertensive subjects than in control subjects (63.6±1.5% and 59.9±1.9% in control and hypertensive subjects, respectively, P<0.05) (). These changes resulted in a marked and significant (P<0.05) increase of the cholesterol/phospholipid ratio in hypertensive subjects (0.37±0.1 and 0.44±0.0 in normotensive and hypertensive subjects, respectively, P<0.05).
fig-ommitted
TABLE 2. Relative Percentages of the Individual Lipid Composition Extracted From Erythrocyte Plasma Membrane of Hypertensive and Control Subjects
Differences were also found in the levels of the fatty acid moieties of phospholipids and cholesterol esters from erythrocyte membranes. Apart from particular changes in the levels of distinct fatty acid species, monounsaturated fatty acids were more abundant in hypertensive subjects, whereas, the content of polyunsaturated fatty acids was higher in blood cell membranes from control subjects ().
fig-ommitted
TABLE 3. Fatty Acid Composition of Phospholipids and Cholesterol Esters in Erythrocytes Membranes (mg/100 mg)
Density of G-Protein Subunits and PKC{alpha}
in the Erythrocyte Membranes of Elderly Hypertensive Subjects
The precise quantification of membrane proteins in normotensive and hypertensive subjects was achieved through quantitative immunoblotting. The correlation between the amount of protein loaded on the gel and integrated optical density (IOD) values was linear in the ranges used. In addition, the steep slope of the curve ensured the reliability of the data. These analyses showed that pertussis toxin–sensitive G-protein {alpha}
-subunit levels were reduced in blood cell membranes of hypertensive subjects. Specifically, the levels of the adenylyl cyclase inhibitory G{alpha}
i1/2 proteins decreased in elderly hypertensive subjects (decreases of 31±10%, P<0.01; ). Similarly, the levels of G{alpha}
o proteins were significantly and markedly reduced in the hypertensive group (decreases of 38±11%, P<0.01; ). However, the decrease observed in the levels of cholera toxin–sensitive G protein (52-kDa G{alpha}
s protein) was not statistically significant in hypertensive subjects (decreases of 16±7%, P>0.05, ). With respect to the common G-protein ß-subunit, the levels of this protein were also downregulated in blood cells of elderly hypertensive patients (decreases of 21±8%, P<0.05, ). Finally, the levels of PKC{alpha}
also showed a marked and significant decrease in the membranes of red blood cells from elderly hypertensive subjects (decreases of 32±8%, P<0.01, ).
fig-ommitted
Figure 1. A, G-protein levels (mean±SEM) in erythrocyte membranes from elderly normotensive (open bars, n=10) and hypertensive (solid bars, n=10) subjects. Levels of G-protein {alpha}
i1/2 (Gi), {alpha}
o (Go), {alpha}
s (Gs), and ß (Gb) subunits are shown. *P<0.05; **P<0.01. B, Representative immunoblots for erythrocyte G proteins from a control (C) and a hypertensive subject (H). About 36 µg of total protein was loaded for both subjects.
fig-ommitted
Figure 2. A, PKC{alpha}
levels (mean±SEM) in erythrocyte membranes from elderly normotensive (control, n=10) and hypertensive subjects (n=10). **P<0.01. B, Representative immunoblot for erythrocyte PKC{alpha}
from a control (C) and a hypertensive subject (H). About 36 µg of total protein was loaded for both subjects.
Discussion
Cells exert an exquisite control of lipid levels both in plasma and organelle membranes.40 The characteristic properties of membranes are dictated by the specific combinations of membrane lipids and proteins that they contain. In this context, the changes in membrane lipid and protein levels in erythrocyte membranes reported here are most probably associated with alterations in cell behavior in elderly hypertensive subjects. These results are of special relevance to the field of cell signaling because a direct relation between membrane lipid levels in human erythrocytes and neurons has been recently discovered.41 Therefore, the changes found in blood cells could reflect alterations in other cell types that control blood pressure.
Different properties of the membrane lipid fraction modulate the activity of membrane proteins. In this context, it has been demonstrated that the membrane cholesterol content (also defined by the cholesterol/phospholipid ratio) regulates the membrane fluidity or microviscosity.42,43 Thus, an increase in this ratio is associated with a decrease in membrane fluidity.43 In addition to cholesterol, the type and abundance of fatty acids (free or esterified) also contribute to the modulation of membrane fluidity.44,45 We found that erythrocyte membranes from hypertensives have a higher cholesterol/phospholipid ratio (0.44±0.0) than those from normotensive subjects (0.37±0.1). This result is in agreement with previous studies showing that the hydrophobic core of erythrocyte membranes is less fluid in hypertensive rats.46 Moreover, we found differences in the fatty acid composition of erythrocyte membranes that may further alter the plasma membrane fluidity in hypertensive subjects. Also, in line with these results, vascular cells of spontaneously hypertensive rats exhibit differences in membrane fatty acids that may contribute to a lower membrane fluidity.47
Previous studies have also associated hypertension with multiple plasma membrane alterations.4,23,24 Since membrane fluidity regulates the activity of several membrane proteins,15,48 the lipid alterations observed in membranes of elderly hypertensive subjects could be involved in altering membrane protein function. Membrane lipids also regulate the formation of hexagonal (HII) phases,16,44 nonlamellar membrane structures that organize into tubular micelles.49–51 HII phases are critical in cells,15,16 where they influence the localization and activity of membrane proteins.15,48 G proteins and PKC are signaling molecules capable of translocating from the cytosol to membranes. They propagate and amplify messages from membrane GPCRs (eg, those which control the blood pressure) to other signaling proteins, and their localization is modulated by the membrane lipid composition and HII-phase propensity.15,16 In the knockouts of the {alpha}
2- and ß1/2-adrenoceptors, it has been shown that these receptor types are implicated in the central and peripheral control of blood pressure, respectively.52,53 These receptors use G{alpha}
i, G{alpha}
o, and G{alpha}
s proteins, further evidence that the results presented here are associated with the control of blood pressure. The levels of G{alpha}
i1/2 and G{alpha}
o were seen here to be markedly and significantly lower in erythrocyte membranes from hypertensive subjects (decreases of 31±10% and 38±11% for G{alpha}
i and G{alpha}
o, respectively, P<0.01), possibly as the result of membrane lipid alterations. Although the common Gß-subunit also appeared to be reduced in hypertensive subjects (21±8% decreases, P<0.05), G{alpha}
s levels were not significantly decreased in hypertensive subjects (16±7%, P>0.05). These alterations in the levels of membrane G proteins could be associated with the reduced G-protein function and/or decreased receptor–G protein coupling described elsewhere, which may alter vasodilator function in hypertensive subjects.25
A reduction in G-protein activity in blood cells of older and hypertensive subjects has been reported previously.29 Although these changes were not accompanied by modulations in whole-cell G-protein levels, they may possibly have been associated with the decreases in membrane G proteins described here. Our study was carried out in cell membranes, so that the levels of G proteins found corresponded to active and/or preactive signal transduction components and may account for alterations in signal propagation. In line with this study and previous works, it has been observed that high blood pressure is associated with downregulation or loss of responsiveness of {alpha}
-adrenoceptors in elderly hypertensive subjects.54
PKC was also found to be reduced in the membranes isolated from hypertensive subjects. As for G proteins, the cellular localization of this signaling enzyme is modulated by the membrane lipid composition and HII-phase propensity.15,48 This enzyme is a key element in many GPCR-related signals, including the control of blood pressure.55 Adrenoceptors (and other GPCRs) can activate PKC through phospholipase C–induced diacylglycerol production. Activated PKC translocates to the plasma membrane,56 where it phosphorylates the serine/threonine residues of a wide variety of proteins, including adrenoceptors and other GPCRs.20,21,57–59 The half-life of activated PKC is less than 1 hour, as the result of its degradation by proteases.60 Therefore, lower PKC levels could either be the result of decreased mRNA expression or increased enzyme activation (and subsequent degradation). The former possibility would be parallel to a decrease in receptor phosphorylation, whereas the latter would be associated with increased receptor phosphorylation and desensitization. Various facts favor the latter hypothesis. First, it has been shown that PKC activation is followed by its degradation, so that protein levels appear to be reduced despite of an elevation in mRNA expression.61,62 Second, an increase in PKC activation would result in increased receptor phosphorylation and reduced adrenoceptor-associated signaling.60 Third, a greater degree of receptor desensitization coupled with lower G-protein levels is in agreement with previous studies demonstrating impaired adrenoceptor signaling. However, this matter requires further investigation because the relevance of this protein in the control of blood pressure and atherosclerosis has also been associated with other signaling events, such as the regulation of endothelin-1, the cytosolic levels of Ca2+, and so forth.63,64 Moreover, it is possible that PKC may exert different or even opposite effects in central and peripheral systems.
In summary, we have found alterations in the levels of erythrocyte membrane lipids in elderly hypertensive subjects that could account for the alterations in the levels of membrane-associated G proteins and PKC also observed here. The decreases in G protein and PKC levels in erythrocyte membranes probably alters GPCR-related signaling in these hypertensive subjects. GPCR-associated signaling alterations could be involved in the etiopathology of hypertension in elderly subjects or may constitute an adaptive mechanism in response to other alterations.
Perspectives
In industrialized countries, cardiovascular pathologies are involved in about one half of all deaths. In fact, if we want to live longer and healthier, it is essential to control cardiovascular, tumoral, and neurodegenerative risk factors. In increasingly aging societies, the knowledge of the molecular alterations underlying high blood pressure in elderly hypertensive subjects and its control are crucial issues. In this study, we highlight some molecular alterations associated with hypertension in elderly subjects. We found that the levels of certain membrane lipids are altered in elderly hypertensive subjects. Specifically, we have shown that the cholesterol/phospholipid ratio and the levels of monounsaturated fatty acids were higher in this group compared with normotensive control subjects, who in turn had lower levels of polyunsaturated fatty acids. We have previously demonstrated that the levels and the types of membrane lipids modulate membrane structure and regulate G protein–membrane and PKC-membrane interactions. When we studied the membrane levels of these proteins, we found significant and marked changes in elderly subjects with high blood pressure. It is feasible that the changes we found in signaling proteins in the membrane were related to alterations in the protein-lipid interactions that resulted from the altered lipid levels. If this hypothesis is correct, it may also be possible to control high blood pressure by modulating the membrane lipid structure. In this context, we have developed molecules capable of acting in this manner, and we have called this therapeutic approach "lipid therapy." We believe that such an approach will become more widespread during the next decades and hence provide many benefits to human health.
Acknowledgments
This work was supported in part by grants FEDER 1FD97–2288 (European Community) and CAO001–002 (Junta de Andalucía) (to V.R.-G.) and by grants FIS00/1029 (Ministerio de Sanidad y Consumo, Spain) and SAF2001–0839 (Ministerio de Ciencia y Tecnología, Spain) (to P.V.E.). We thank the Marathon Foundation for its financial support. RA is a Ramón y Cajal Fellow. We are grateful to the director, nurses, and medical personnel of the Residencia de la Tercera Edad Heliópolis for their cooperation during the period of the study.
Received June 14, 2002; first decision July 10, 2002; accepted November 7, 2002.
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(Andrei Forclaz; Marc Maillard; Jürg Nussberger; Hans R. Brunner; Michel Burnier)
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