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748 Lipid Bilayer in Genetic Hypertension Anna F. Dominiczak, Dan F. Lazar, Arun K. Das, and David F. Bohr Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Membrane microviscosity, phospholipid composition, and turnover were measured in cultured vascular smooth muscle cells isolated from mesenteric arteries of stroke-prone spontaneously hypertensive and age-matched, normotensive Wistar-Kyoto rats. Membrane microviscosity, measured with fluorescence polarization, revealed greater microviscosity (lower fluidity) of the membranes isolated from smooth muscle cells from hypertensive as compared with those isolated from normotensive rats (p<0.01). Preincubation of membranes from hypertensive rats with 5 mM calcium reduced membrane microviscosity in "core" and in "surface" regions of the bilayer toward values observed in Wistar-Kyoto rats. Phospholipid composition did not differ between intact aortas and cultured mesenteric cells or between those tissues obtained from normotensive and from hypertensive rats. The total lipid-associated radioactivity was significantly lower in cells from stroke-prone spontaneously hypertensive rats than in those from Wistar-Kyoto controls (/?<0.01). Phosphatidylcholine incorporated 70% and phosphatidylinositol 16% of total lipid-associated radioactivity, with no difference between cells from hypertensive and normotensive animals. Turnover of phosphatidylethanolamine was greater in cells from Wistar-Kyoto rats (p=0.02), whereas turnover of phosphatidylserine was greater in cells from stroke-prone spontaneously hypertensive rats (p=0.04). The greater microviscosity of the lipid bilayer in hypertension is a generalized defect of the matrix in which the transport proteins function. We hypothesize that this defect is responsible for the multiple abnormalities of membrane transport systems that have been described in genetic hypertension. (Hypertension 1991;18:748-757) H ypertension has been associated with abnormalities in many membrane transport systems including channels for sodium and potassium1"3 and calcium,4 exchangers for sodium/ hydrogen56 and for sodium/calcium,7 as well as active pumps for sodium/potassium78 and for calcium.910 The diversity of the membrane proteins that have been found to have abnormal functions in hypertension led to the hypothesis that this generalized dysfunction of diverse protein transport systems is caused by a fault in the matrix in which they all function, the lipid bilayer.11 There is extensive evidence that the functions of membrane proteins are influenced by properties of the lipid bilayer.12 For example, Remmers et al13 have observed that the opioid receptor can be modulated by specific fatty acids through mechanisms From the Departments of Physiology (A.F.D., D.F.B.), Biological Chemistry (D.F.L.), and Neuroscience Laboratory (A.K.D.), University of Michigan, Ann Arbor, Mich. Supported by grant HL-18575 from the National Institutes of Health. This work was done during the tenure of the British American Research Fellowship (to A.F.D.) supported by the British Heart Foundation and American Heart Association. Address for correspondence: Dr. A.F. Dominiczak, MRC Blood Pressure Unit, Western Infirmary, Glasgow, Gil 6NT, Scotland, UK. Received March 12, 1991; accepted in revised form August 16, 1991. involving membrane fluidity. In addition, the coupling of the /3-adrenergic receptor to adenylate cyclase is enhanced by decreasing membrane microviscosity,14 and Na+, K+-ATPase activity is accelerated when the membrane microviscosity is low.15 Fluorescence polarization studies in erythrocytes and platelets from patients with essential hypertension and from spontaneously hypertensive rats (SHR) showed increased microviscosity as compared with normotensive controls.16-19 Tsuda et al,20 using an electron spin resonance and spin-labeling method, confirmed decreased membrane fluidity in erythrocytes and cultured vascular smooth muscle cells (VSMC) of SHR. The physicochemical properties such as microviscosity of biomembranes are affected by the content and physiological states of cholesterol, phospholipids, and their fatty acids.15 Okamoto et al21 have shown that renal membrane phospholipids, especially phosphatidylcholine and phosphatidylethanolamine, decrease with age in SHR. This decrease is associated with increased phospholipase A2 activity and phospholipid degradation. The majority of studies of lipid bilayer composition and its functional properties in hypertension have been performed on circulating blood cells. There is very little information on lipid bilayer in VSMC. This tissue is relevant since it is directly involved in the Dominiczak et al TABLE 1. Data on Blood Pressure, Weight, and Age of Rats Variable n Blood pressure (mm Hg) Weight (g) Age (days) SHRSP WKY 40 40 122±4* 104 ±6 45 ±5 88±3 111±9 44±4 SHRSP, stroke-prone spontaneously hypertensive rats; WKY, Wistar-Kyoto rats. */?<0.001. increased vascular reactivity and medial hypertrophy, hallmarks of the hypertensive process. The purpose of this study is to compare membrane microviscosity, phospholipid composition, and metabolism in VSMC from stroke-prone SHR (SHRSP) and from Wistar-Kyoto normotensive rats (WKY). Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Methods Animals SHRSP and WKY rats were obtained from colonies in the Department of Anatomy and Cell Biology at the University of Michigan. These are inbred colonies, derived from a stock supplied by the National Institutes of Health (NIH), Bethesda, Md. Data on age, weight, and blood pressure of rats are given in Table 1. Rats were decapitated and the mesenteric arteries and thoracic aortas were removed under aseptic conditions and were placed in ice-cold Hanks' balanced salt solution (HBSS). Aortas (five per preparation) were cleaned of fat and adventitia and immediately were frozen by placing in aluminum foil over a mixture of dry ice and acetone. Aortas were stored in -70°C before the lipid extraction procedure. Isolation and Culture of Rat Mesenteric Artery Smooth Muscle Cells VSMC were harvested from enzymatically dissociated rat mesenteric arteries by modifications of the methods described by Gunther et al22 and Lyall et al.23 All procedures were carried out under aseptic conditions. The superior mesenteric artery with its major branches was excised, en bloc, from its origin at the aorta to the mesenteric border of the intestine and was placed in a petri dish containing ice-cold HBSS with 0.2 mM added Ca2+. Fat, adventitia, and veins were removed by blunt dissection, the branches were cut off close to the main trunk of the superior mesenteric artery, and the artery was cut open lengthwise to expose the endothelial layer. The cleaned mesenteric arteries (five per preparation) were transferred into a petri dish containing 5 ml enzyme dissociation mixture: HBSS with 0.2 mM added Ca2+, 1.25 mg/ml collagenase, 0.25 mg/ml elastase, 0.5 mg/ml soybean trypsin inhibitor, and 2 mg/ml bovine serum albumin. The arteries were incubated at 37°C in a humidified 5% CO2-95% air atmosphere for approximately 30 minutes. Then the arteries were transferred to fresh HBSS and tritu- Lipid Bilayer in Hypertension 749 rated 10 times through a plastic Pasteur pipette. They were cut into small pieces, transferred to fresh enzyme dissociation mixture, and incubated at 37°C with periodic trituration until a single cell suspension was obtained (usually 45-60 minutes). The tissue suspension was sieved through 106-^.m stainless steel mesh to separate dispersed cells from undigested vessel wall fragments and debris. The filtered suspension was diluted in 20 ml Dulbecco's modified Eagles medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, 10% (vol/vol) equine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 jig/ml streptomycin and then was centrifuged (400g, 5 minutes). The cell pellet was resuspended in 5 ml DMEM supplemented as described above. The dispersed cells were plated in a 25-cm2 culture flask and were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. After the first 24 hours, the medium was replaced to remove nonadherent cells. Plating efficiency was consistently above 50%. Medium was changed at 48-72hour intervals. VSMC had usual growth characteristics, took 7 days to reach confluence after plating, and at confluence, had the typical "hill and valley" pattern.22 Cells up to the 10th passage were harvested once a week with trypsin-EDTA and passaged at a 1:3 ratio in 162-cm2flasks.Before experiments, cells were deprived of serum by a 24-48-hour incubation in fresh DMEM supplemented with 0.4% fetal calf serum. The cells were positively identified as smooth muscle by indirect immunofluorescent staining for a-actin. To detect the presence of smooth muscle-specific a-actin, the cells grown to 50% confluence on sterilized round cover slips, 12-mm diameter, werefixedin acetone: methanol (1:1, vol/vol) for 2 minutes at -20°C. They were then incubated with monoclonal anti-a-smooth muscle actin antibody diluted 1:400 in phosphate-buffered saline (PBS) with 1% bovine serum albumin for 60 minutes at 37°C. After washing three times in PBS of the same composition, the cells were incubated with secondary antibody-anti-mouse immunoglobulin G (IgG) (whole molecule)fluoresceinisothiocyanate (FITC) conjugate in the dark for 45 minutes at 37°C. After being washed three times in PBS, cover slips were mounted onto microscope slides on glycerol 9:1 (vol/vol) in PBS and examined under afluorescencemicroscope at a magnification of x400 and x630. NIH-3T3 cells (mesenchymal cell line; gift from Dr. Donald MacCallum) and VSMC incubated with secondary antibody only were used as controls. An example of the immunofluorescence study of mesenteric cells at the stage of second passage is shown in Figure 1. Membrane Preparation Confluent monolayers of VSMC were washed three times with ice-cold HBSS (Ca2+, Mg2+-free formula) and detached by gentle scraping with the aid of a "rubber policeman." The suspension was centrifuged for 5 minutes at 400g, and the pellet was suspended in hypotonic buffer of the following composition (mM): Na2HPO4 0.61, KH2PO4 0.38, MgSO4 750 Hypertension Vol 18, No 6 December 1991 FIGURE 1. Photomicrograph of mesenteric smooth muscle cells that reacted with anti-a smooth muscle actin antibody followed by an anti-mouse immunoglobulin G fluorescein isothiocyanate conjugate as described in "Methods." Magnification, x 1,800. Bar (1 cm) =5 fim. Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 0.2, dithiothreitol 1, pH 7.4. The pellet was then dispersed in a Dounce homogenizer and was centrifuged for 15 minutes at 20,000g, 4°C. The resulting pellet was resuspended in 50 mM Tris HC1, pH 7.4, at a protein concentration of 1 mg/ml. Determination of Protein The methods used were according to Lowry et al24 and Bradford25 with bovine serum albumin as a standard. The membrane protein was solubilized initially with 1M NaOH- for 30 minutes at 25°C. Measurement of Membrane Microviscosity A previously described method13 was applied, except in our study both fluorophores were measured at the same wavelengths for excitation and emission, respectively. The fluorescent probes diphenylhexatriene (DPH) (solution in Af,JV-dimethylformamide) and its charged trimethylammonium derivative (TMA-DPH) (solution in tetrahydrofuran) were incubated with membranes at 25°C for 30 minutes and 60 minutes, respectively. The molar ratio of probe to membrane phospholipid was approximately 1:200 in all experiments. It was determined that this ratio of probe to membrane phospholipid did not perturb membrane microviscosity. Fluorescence polarization was measured in a spectrofluorometer (SLM Instruments, Inc., Urbana, 111.) at wavelengths of 340 nm for excitation and 450 nm for emission. The results are expressed as anisotropy units (r), where r=(lfl-lgfl)(lfl+2190) and IQ and 1% represent the intensities of light when polarizers were in parallel or perpendicular orienta- tion, respectively. Differences in the efficiency of transmitting vertically and horizontally polarized light were corrected for by determining the polarization ratio (l0—lgo) using horizontally rather than vertically polarized excitation light. The advantage of expressing fluorescence polarization as anisotropy is the additive nature of the latter.13 Phospholipid Composition of Cultured Vascular Smooth Muscle Cells and Intact Aortas Confluent monolayers of VSMC were washed three times with ice-cold physiological salt solution (PSS) of the following composition (mM): NaCl 130, KC1 4-7, KH2PO4 1.18, MgSO4-7H2O 1.17, CaCl2-2H2O 1.6, NaHCO314.9, dextrose 5.5, CaNa2-EDTA 0.026. The cells were scraped into 15-ml ice-cold PSS and were centrifuged at 400g for 5 minutes. Lipid extraction was performed as described by Lee and Hajra.26 In brief, cell pellets and intact aortas were homogenized three times for 30 seconds each at 2.5 setting, in chloroform: methanol (1:1, 12 ml/g of tissue) with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, N.Y.). The homogenate was centrifuged (l,000g, 10 minutes), and the supernatant was saved. The residue was dispersed in water (0.5 ml/g tissue) by agitation in a sonic bath, and this dispersed residue was rehomogenized in chloroform: methanol (1:1, 6 ml/g original tissue). This second homogenate was centrifuged (l,000g, 10 minutes), and the supernatant was combined with the first supernatant. To the combined supernatants, an additional amount of chloroform was added (9 ml/g tissue) and mixed well, and 0.9% aqueous NaQ (5.5 ml/g tissue) was added to the extract to facilitate the separation. The mixture was vortex-mixed Dominiczak et al Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 and centrifuged (l,000g, 5 minutes), and the upper layer was removed. The lower layer was dried under a stream of nitrogen at 40°C and resuspended in 100 fi\ chloroform: methanol (2:1, vol/vol). The phospholipids were resolved by monodimensional thin-layer chromatography (TLC) using the following solvent systems: 1) chloroform:methanol:0.25% aqueous KC1:ethyl acetate: isopropanol (60:18:12:36:50, vol/vol); 2) chloroform : methanol: acetic acid:H2O (100:40:12:4, vol/ vol); and 3) chloroform: methanol: 40% methylamine (63:35:10, vol/vol). The individual phospholipids, cochromatographed with standards and visualized with primuline, were identified and scraped for phosphate determination performed according to Ames.27 Samples of scraped phospholipids and K2HPO4 standards were mixed with 10% Mg(NO3)2 in 95% ethanol, dried and heated in a flame until brown vapor disappeared. After cooling, 0.5N HC1 (0.3 ml) was added, and samples were heated in a heating block at 100°C for 15 minutes. This was followed by incubation with 0.7 ml of a mixture of 10% ascorbic acid: 0.42% ammonium molybdate in IN H2SO4 (1:6, vol/vol) for 30 minutes at 45°C. The concentration of phosphate was determined spectrophotometrically (Beckmann, DU70, Arlington Heights, 111.) at 820 nm. The results are expressed as a percentage of total phospholipid content in each lipid extract. Isolation of Platelets Washed platelets were prepared as described previously.28 Briefly, platelet-rich plasma was prepared by centrifuging citrated (1 ml of 3.9% [wt/vol] sodium citrate/9 ml of blood) whole blood at 400g for 5 minutes at room temperature. EGTA (5 mM) was added to the platelet-rich plasma, which was centrifuged at 40Qg for 10 minutes. The supernatant was removed, and the pellet was suspended in calciumpoor medium containing (mM) NaCl 130, KC1 4.7, MgCl2 1, glucose 10, HEPES 20, pH 7.4. The platelets were counted in a Coulter counter (Coulter Corp., Hialeah, Fla.) and the concentration of platelets adjusted to 1.5-2xlO8/ml. Lipid Bilayer in Hypertension 751 solvent system of chloroform: methanol: 40% methylamine (63:35:10, vol/vol). Individual phospholipids were localized by overnight (12-14 hours) autoradiography (Kodak X-OMAT film), and 32P incorporation was quantified by scraping the phospholipid spots from the silica plate and counting in the scintillation counter. Statistical Analysis Data are presented as the mean±SEM of n experiments (five animals were used for each tissue/cell culture preparation). Statistical analysis was performed using the unpaired Student's t test with Bonferroni correction for multiple comparisons. A value ofp < 0.05 was regarded as a significant difference. Materials Phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, collagenase (type II), elastase (type IV), soybean trypsin inhibitor (type I-S), fetal bovine serum, equine serum, monoclonal anti-asmooth muscle actin, and anti-mouse IgG (whole molecule) FITC conjugate were obtained from Sigma Chemical Co., St. Louis, Mo. DPH and its charged derivative TMA-DPH were purchased from Molecular Probes, Junction City, Ore. Carrier-free 32P (1 mCi/ml) was bought from Amersham Corp., Arlington Heights, 111. DMEM, penicillin, streptomycin, glutamine, trypsin-EDTA, and HBSS (Ca2+, Mg2+-free formula) were obtained from Irvine Scientific, Santa Ana, Calif.; 20x20 cm TLC plates (silica gel 60, 0.25 mm thick) were purchased from Merck, Cherry Hill, N.J. All other chemicals were of analytical grade. Results Membrane Microviscosity in Vascular Smooth Muscle Cells Measurements with the membrane "core" probe DPH and "surface" probe TMA-DPH revealed sigLabeling of Phospholipids in Vascular Smooth Muscle nificant difference in microviscosity between memCells and Platelets branes obtained from SHRSP cells and those obVSMC were trypsinized and pelleted at 400g for 10 tained from WKY cells (Figure 2). This difference minutes. The pellet was resuspended in HEPES was slightly greater for core than for surface probe buffer of the following composition (mM): NaCl 130, measurements. Preincubation of VSMC membranes KC1 4.7, MgCl2 1, glucose 10, HEPES 20, CaCl2 1.6, with 5 mM calcium, added to Tris buffer 30 minutes pH 7.4. Aliquots of the VSMC (or platelet) suspenbefore fluorescent probes, significantly reduced sion (0.58 ml each) were incubated with 0.02 mCi membrane microviscosity in VSMC from SHRSP for phosporus-32 for 0, 30, 60, 120, and 180 minutes at DPH and TMA-DPH measurements. Membrane mi37°C in a shaking water bath. Platelets were incucroviscosity in VSMC from WKY was also slightly bated in calcium-poor medium as described above. reduced, but only the value for TMA-DPH reached The reaction was terminated by addition of 2.25 ml statistical significance (Figure 2). chloroform: methanol (1:2, vol/vol). The phospholipids were extracted under acidic conditions according Phospholipid Composition to the method of Bligh and Dyer (as modified by LaBelle and Hajra29). The lower layer was evapoPhosphatidylcholine was the major phospholipid in rated to dryness under a stream of nitrogen at 40cC, cultured vascular smooth muscle cells and in intact and phospholipids were separated by TLC using aortas (approximately 40% of total phospholipid con- 752 Hypertension Vol 18, No 6 December 1991 SHRSP ft WKY • TMA DPH Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 FIGURE 2. Bar graph shows microviscosities of the lipid bilayer from cultured vascular smooth muscle cells from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP); effect of 5 mM calcium. Measurements were made using fluorescent probes: diphenylhexatriene (DPH) for the core of the bilayer and the charged trimethylammonium derivative (TMA-DPH) with which the microviscosity of the surface region is monitored. Data are mean±SEM from seven studies comparing cells from SHRSP and WKY and from four studies in 5 mM Ca2+. **p<0.01 SHRSP vs. WKY and SHRSP vs. SHRSP in 5 mM Ca2+; *p<0.05 WKY vs. WKY in 5 mM Ca2+. tent). No significant differences were observed in phospholipid composition between mesenteric VSMC and intact aortas. There was also no difference between vascular tissue or cells obtained from SHRSP and those obtained from WKY (Figure 3). 180 120 60 30 30 180 FIGURE 4. Autoradiogram of thin-layer chromatogram in which the phospholipids of cultured vascular smooth muscle cells had been labeled by incubation with phosphorus-32 for 30, 60, 120, or 180 minutes. Labeling of phospholipids from the Wistar-Kyoto rat (WKY) cells is greater than that of stroke-prone spontaneously hypertensive rat (SHRSP) cells. Discrete spot above phosphatidylethanolamine probably represents diphosphatidylglycerol (cardiolipin); it accounted for less than 1% of total radioactivity. O indicates the origin; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. ity of each spot was determined by scraping and scintillation counting. VSMC from SHRSP showed less 32P labeling of phosphatidylcholine and phosphatidylethanolamine as compared with 32P labeling of the same phospholipids from WKY cells (Figures 5B 32 Phosphorus-32 Labeling of Phospholipids in Vascular and 5C). Incorporation of P into phosphatidylinositol and phosphatidylserine also tended to be lower Smooth Muscle Cells in cells from SHRSP than in those from WKY, but 32 A representative autoradiogram showing P labelthese differences did not reach statistical significance ing of phospholipids in VSMC is shown in Figure 4. at any time point (Figure 5D). After chromatographic separation, the 32P radioactivThe total lipid-associated 32P radioactivity was significantly lower in cells from SHRSP than in those from WKY after 60, 120, and 180 minutes of incubation (Figure 5A). The deficit in total lipid-associated 32 P observed in VSMC from SHRSP indicates that this tissue has a generalized defect in its mechanism for incorporating exogenous phosphate into lipids. To determine whether there were also differences among the phospholipids in their uptakes of 32P, the results for individual phospholipid classes were expressed as percentages of total lipid-associated radioactivity after 2 hours of incubation (Table 2). The phosphatidylcholine incorporated approximately 70% of the 32P, and there was no significant SPh PA PC PE PI difference in 32P labeling between cells from SHRSP and WKY (Table 2). Phosphatidylinositol incorporated FIGURE 3. Bar graph shows phospholipid composition of approximately 16% of the 32P, and again no significant vascular smooth muscle cells (VSMC) and aortas. Values difference was observed between the two cell types. represent mean±SEM for eight studies on VSMC and four Phosphatidylethanolamine that incorporated relatively studies on aortas in each group. No difference was found much less radiolabeled phosphorus retained the origieither between VSMC and aortas or between these tissues from nal differential labeling with cells from WKY incorpostroke-prone spontaneously hypertensive rats (SHRSP) and rating 6.2±0.7% 32P into phosphatidylethanolamine Wistar-Kyoto rats (WKY). PI, phosphatidylinositol; PA, phosand cells from SHRSP incorporating 4 ±0.4% 32P into phatidic acid; PS, phosphatidylserine; SPh, sphingomyelin; phosphatidylethanolamine (p=0.02; Table 2). In conPC, phosphatidylcholine; PE, phosphatidylethanolamine. Dominiczak et al Lipid Bilayer in Hypertension 110 •o 110 110 753 ISO Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 i 120 100 0 SO 120 TIME(mln) 110 TIHE(mln) FIGURE 5. Line graphs show time course of phosphorus-32 incorporation in phospholipids of vascular smooth muscle cells from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). PL, total phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. Rate of incorporation of 32P into each phospholipid from WKY is faster than in that from SHRSP. Each value is the mean±SEMfor six or eight determinations. *p<0.05; **p<0.01. trast, labeling of phosphatidylserine showed slightly but significantly more 32P in this phospholipid class extracted from cells obtained from SHRSP than in those obtained from WKY (11.3±1.3% and 7.8±0.8%, respectively;/? =0.04, Table 2). TABLE 2. PE VSMC (% total lipid radioactivity at 2 hrs) # 1 WKY 70.4±1.9 6.1 7.4 SHRSP 67.7±1.7 10.1 11.1 1.0 Strain WKY SHRSP PS WKY PIP2 Platelets #2 6.2±0.7 1.1 4.0±0.4* 1.1 1.6 7.8 ±0.8 7.3 12.2 11.3±1.3* 11.8 12.1 WKY 16.0±1.9 67.0 51.7 SHRSP 16.9±1.0 SHRSP PI Related comparisons of 32P uptake in WKY and SHR have been made previously on platelets.30-31 We Phosphorus-32 Labeling of Phospholipids in Smooth Muscle Cells and Platelets Phospholipid PC Phosphorus-32 Labeling of Phospholipids in Platelets From SHRSP and WKY 62.6 53.6 WKY 18.1 27.7 SHRSP 14.3 21.6 Results for vascular smooth muscle cells are mean±SEM of eight studies; data for platelets represent two single experiments. VSMC, vascular smooth muscle cells; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PIP 2 , phosphatidylinositol 4,5-bisphosphate; WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats. V<0.05 WKY vs. SHRSP. 754 Hypertension Vol 18, No 6 December 1991 SHRSP WKY PE PC • * mm 120 60 I PS PI 30 I PIP2 o 30 60 120 Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 FIGURE 6. Autoradiogram of thin-layer chromatogram in which the phospholipids of platelets from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) had been labeled with phosphorus-32 for 30, 60, or 120 minutes. O, indicates the origin. Phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were all labeled in platelets from both strains of rats. Spot identified as PIP2 might contain lyso PI as complete separation was not possible. therefore repeated studies as we had done on VSMC in two experiments on platelets. Results of these two experiments were similar (Table 2). The autoradiogram of one such experiment is presented in Figure 6. Spots unambiguously identified as phosphatidylcholine were present on autoradiograms of platelet phospholipids from SHRSP and WKY. The 32P incorporation into phosphatidylcholine was greater in platelets from SHRSP than in those from WKY (Figure 6 and Table 2). The pattern of 32P uptake by the individual phospholipids in platelets is entirely different from that in VSMC. This difference is evident in Table 2 in which 32P uptake of the two platelet experiments is compared with the mean uptake values for eight VSMC studies. The uptake value for each phospholipid is expressed as the percentage of total lipid-associated radioactivity. Discussion The major finding of this study is that membrane microviscosity, as measured with DPH the core or TMA-DPH the surface probe, was significantly greater in membranes from cultured VSMC obtained from SHRSP than in those obtained from the WKY normotensive control strain. Moreover, membrane microviscosity was modified in vitro by incubating the membranes with 5 mM calcium. This treatment significantly lowered microviscosity of SHRSP membranes in core and surface regions of the bilayer, making these membranes very similar to those obtained from VSMC from normotensive controls. Early studies on membrane microviscosity in genetic hypertension were limited to membranes of circulating blood cells (red blood cells and platelets) and to measurements with the membrane core probe DPH. Studies with red blood cells involved only the plasma membrane, whereas in our studies with VSMC, both plasma membrane and intracellular membranes were isolated. Nevertheless, these earlier studies of red blood cells of SHR1617-32 or of patients with essential hypertension1819 demonstrated increased microviscosities of the same order of magnitude as that in our current data. Devynck et al33 reported similar changes in microviscosity in the plasma membranes of hepatocytes, synaptosomes, and cardiomyocytes of SHR. Tsuda et al,20 using an entirely different methodology, an electron spin resonance found that values of outer hyperfine splitting and of the order parameter determined from electron spin resonance spectra for fatty acid spin-label agent (5-nitroxy stereate) were significantly higher in red blood cells and cultured VSMC from SHR than in those from WKY rats. These data were interpreted as indicating a decreased membrane fluidity in the SHR. The same group34 showed that the red blood cells pretreated with the calcium ionophore A23187 and 1 mM calcium chloride had increased microviscosity as compared with the untreated cells. These changes were more prominent in the red blood cells from hypertensive rats and patients with essential hypertension than in their respective normotensive controls. The authors argued that an abnormality of cellular calcium handling might affect physicochemical properties of the biomembranes in hypertension.34 The current study is the first fluorescence polarization study showing increased microviscosity of VSMC membranes from genetically hypertensive rats. Our findings demonstrate that the microviscosity of the hydrophobic core of the lipid bilayer of cultured VSMC from genetically hypertensive rats is greater than that of these cells from normotensive controls. This is the first experimental evidence indicating that there is a similarly greater microviscosity in the surface region of the bilayer of any cell isolated from genetically hypertensive rats. The hydrophilic surface of the bilayer consists of the polar heads of the phospholipids, and in all membranes it has a greater viscosity (lower fluidity) than does the central core, which consists of the acyl chains. Our observation that the microviscosity is greater in both the surface and the core regions suggests that the abnormality of the lipid bilayer in hypertension is generalized and therefore has the potential for affecting the function of transport proteins. In line with these findings is the frequently reported observation that membrane permeability to Na+, K+, and Ca2+ is increased in hypertension.1-4 The possibility that the increased microviscosity may be the cause of these greater cation fluxes in hypertension is supported by observations of the effects of increased extracellular calcium concentration on membrane microviscosity and on cation fluxes. In the current study, we observed that an increase in calcium concentration decreases microviscosity of the lipid bilayer of VSMC from SHRSP. Dominiczak et al Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Jones and Hart35 and Furspan and Bohr3 studied the effects of the same magnitude of increase in extracellular calcium concentration on potassium efflux from vascular smooth muscle and from lymphocytes, respectively. They found that the increase in extracellular calcium concentration caused a decrease in potassium efflux (i.e., calcium affected the membrane in such a way that the protein channel responsible for potassium efflux became less active). Their studies demonstrated that a concentration of 5 mM calcium had clear depressing effects on monovalent cation fluxes and on vascular reactivity. We therefore chose this concentration (5 mM) for our study of the effect of calcium on membrane microviscosity. These observations suggest that under conditions of hypertension or high extracellular calcium, membrane microviscosity may have an effect on cation fluxes. The increased microviscosity of hypertension is accompanied by an increase in cation fluxes, whereas when microviscosity is reduced by increasing extracellular calcium, the cation fluxes are depressed. Bialecki and Tulenko36 have studied the effects of another intervention and reported observations that give further support to this relation between membrane microviscosity and permeability. They observed that increased incorporation of cholesterol into VSMC membrane caused an increase in membrane permeability to calcium. Cholesterol is known to increase membrane microviscosity.3738 Thus, cholesterol, which increases microviscosity, causes an increase in permeability as in hypertension; whereas calcium, which reduces microviscosity, causes a decrease in permeability to levels similar to those of normotensive rat. Data from two laboratories documented that the cell membrane of various tissues from hypertensive humans and from the SHR binds less calcium than do tissues from normotensive controls.3339-41 The molecular basis for this abnormality might be related to deficiency in calcium binding proteins or abnormalities of negatively charged phospholipids. Kowarski et al42 have reported that various tissues from SHR contain less of an integral calcium binding protein than those from WKY. However, the affinity of this protein for calcium is too high: the binding sites would be saturated at physiological concentrations of extracellular calcium. This leaves the phospholipids of the lipid bilayer, particularly the negatively charged phosphatidic acid, phosphatidylserine, and phosphatidylinositol as the most important components that bind calcium, especially at low affinity sites.38-43 Contrary to our observation that membrane microviscosity is decreased by an elevated calcium concentration, several investigators have reported that calcium causes an increased microviscosity.44-46 For example, Sauerheber et al44 reported that incubation of rat epididymal adipocyte ghosts with calcium chloride decreased membrane fluidity in a concentration-dependent manner. We have no clear explanation for the difference between these results and ours except the very different cell type studied and Lipid Bilayer in Hypertension 755 different methodology of fluidity measurements: whole cell electron spin resonance versus membranes and polarization spectrofluorometry. The interaction of calcium with the membrane has been shown to affect phase separation, bilayer-nonbilayer transition, changes in membrane permeability, and fusion.37-38 Calcium may also provide bridging between proteins and lipids, which could exert a marked effect on the membrane assembly.38 It is clear that there are many tenable alternatives to our speculation relating calcium "stabilization" to bilayer microviscosity. We carried out studies to determine whether these clear differences in membrane microviscosity and permeability between VSMC of WKY and SHRSP could be caused by differences in phospholipid composition or their dynamic properties within the lipid bilayer. Direct measurements of phospholipid composition in cultured VSMC and in intact aortas demonstrated a similarity between the two vascular preparations, suggesting that this property of the membrane had not undergone a phenotypic change in culture. There was no difference in the content of major classes of phospholipids between VSMC from SHRSP as compared with those from WKY rats. This is in agreement with data on phospholipid composition in platelets30 and thoracic aortas47 from SHR and WKY. The authors of the latter study measured only phosphatidylethanolamine and phosphatidylcholine content. Okamoto et al21 presented evidence for increased activity of phospholipase A2 in renal membranes of SHRSP as compared with WKY. This phospholipase activity increased with age in SHRSP and was associated with parallel decreases of phosphatidylcholine, phosphatidylethanolamine, and of the phosphatidylcholine-to-sphingomyelin ratio. Phosphatidylcholine and phosphatidylethanolamine act as membrane fluidizers, whereas proteins, cholesterol, and sphingomyelin act as rigidifiers.37 Okamoto et al21 speculated therefore that the renal membrane of SHRSP became "rigid" with age due to the changes in phospholipid composition. The method used in the current study permitted the determination of phosphate content with precision to 0.01 /umol,27 and no difference was observed between those measurements in VSMC from WKY and from SHRSP. It should be stressed, however, that under physiological conditions, lipid constituents of biological membranes diffuse freely in the plane of the membrane. Another feature that adds to this complexity is the asymmetric distribution of phospholipids in the endofacial and the exofacial sides of the membrane.37 It might therefore be more relevant, for pathophysiological considerations, to measure lipid microviscosity rather than the chemical composition of the phospholipids within the bilayer. Remmal et al30 and Marche et al31 presented evidence for enhanced turnover of phosphatidylcholine in platelets of SHR, SHRSP, and inbred Dahl salt-sensitive rats as compared with their respective normotensive control strains. In contrast, platelets from deoxycorticosterone acetate (DOCA)-salt 756 Hypertension Vol 18, No 6 December 1991 Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 hypertensive rats showed similar 32P phosphatidylcholine labeling as their normotensive controls. It was argued that the enhanced turnover of the most abundant membrane phospholipid may constitute a primary membrane alteration, ultimately resulting in increased free cytosolic calcium and increased sensitivity to agonists.31 The current experiments, examining 32P labeling of individual phospholipids in VSMC, gave results that differed from those obtained in platelets.3031 VSMC from both sources exhibited a major radioactive spot identified as phosphatidylcholine. The total lipidassociated radioactivity was greater in VSMC from WKY than in those from SHRSP. The possible role of the different metabolic pools of ATP or lack of the isotopic equilibrium with the y-phosphate of the ATP, or both, cannot be excluded as the basis for this difference. Insight into differences that may occur between WKY and SHRSP phospholipids can be obtained by expressing the 32P labeling of the individual phospholipids as a percentage of total lipidassociated radioactivity after incubation with the isotope.3048 After 2 hours of incubation, phosphatidylcholine labeling was similar in cells from SHRSP and WKY, whereas 32P incorporation into phosphatidylethanolamine was significantly greater in cells from WKY than in those from SHRSP. Phosphatidylethanolamine has been shown to act as membrane fluidizer37 and to affect the activity of Ca2+ATPase possibly through interactions between the bilayer surface and the extramembranous portions of the calcium pump protein.15 There was also a small but significant difference in 32P labeling of phosphatidylserine, which similar to phosphatidylethanolamine, is located in the inner half of the bilayer. It might be relevant that the calcium phospholipid-dependent protein kinase C requires phosphatidylserine for activation and that mesenteric arteries from SHRSP have been shown to be more responsive to the contractile effects of protein kinase C activators than those from WKY.49 The observed discrepancies in 32P labeling between VSMC in this study and platelets30-31 may be related to individualities of the cell types. However, because of this difference in labeling between VSMC in our laboratory and platelets as studied by Marche et al,31 we felt that it was incumbent on us to repeat the platelet studies in our laboratory. Marche observed that after 2 hours of incubation with 32P, platelets from SHRSP but not those from WKY exhibited radioactive spots identified as phosphatidylcholine. In contrast to this observation, our autoradiograms showed radioactive spots identified as phosphatidylcholine in platelets from SHRSP and WKY at each time point studied (see Figure 6). Counting of the radioactivity in these spots confirmed Marche's observation that more 32P was incorporated into phosphatidylcholine in SHRSP, but the differences observed in the current study were less pronounced than the 10- to 16-fold difference reported previously.31 The explanation for these discrepancies is not clear; the possibilities to consider are: 1) betweenstrain differences in genetically hypertensive rats obtained from commercial suppliers as compared with inbred colonies used in this study50 and 2) time of exposure of the x-ray film to TLC plates; the total lipid-associated radioactivity per microgram of protein tends to be low in platelets and longer exposure (up to 48 hours) is needed to visualize the phosphatidylcholine in platelets from WKY. In conclusion, this is the first fluorescence polarization study to establish that membrane microviscosity of VSMC is elevated in SHRSP as compared with membranes from WKY. It confirms observations on membranes from red blood cells and platelets suggesting that this bilayer abnormality may be generalized. More importantly, the current observation was made in a tissue that is involved directly in vascular contractility and thus in regulation of blood pressure. Our observations also demonstrate for the first time that the abnormality affects similarly the core and the surface regions of the lipid bilayer. In addition, we observed that an increase in calcium concentration causes a decrease in membrane microviscosity in both regions. Since an increase in extracellular calcium concentration also has been shown to decrease membrane permeability, we hypothesize that the increased microviscosity of the cell membrane in hypertension causes the increase in membrane permeability to K+, Na+, and Ca2+ observed in hypertension. Acknowledgments We thank Dr. Fedor Medzihradsky and Dr. Amiya K. Hajra for sharing their insight into lipid biochemistry. 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Hypertension 1990; 16:43-48 48. Tysnes OB, Verhoeven AJM, Holmsen H: Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets. Biochim Biophys Ada 1986;889:183-191 49. Turla MB, Webb RC: Enhanced vascular reactivity to protein kinase C activators in genetically hypertensive rats. Hypertension 1987;9(suppl III):IH-150-III-159 50. Kurtz TW, Castro R, Simonet L, Printz MP: Biometric genetic analysis of blood pressure in the spontaneously hypertensive rat. Hypertension 1990;16:718-724 KEY WORDS • vascular smooth muscle • cell culture • blood platelets • membranes • phospholipids • membrane fluidity • spontaneously hypertensive rats Lipid bilayer in genetic hypertension. A F Dominiczak, D F Lazar, A K Das and D F Bohr Hypertension. 1991;18:748-757 doi: 10.1161/01.HYP.18.6.748 Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/18/6/748 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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