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(Circulation Research. 1996;79:974-983.)
© 1996 American Heart Association, Inc.

Articles

Selectively Enhanced Cellular Signaling by G_i Proteins in Essential Hypertension

G_i2, G_i3, Gß₁, and Gß₂ Are Not Mutated

Frank Pietruck, Albrecht Moritz, Michael Montemurro, Alexandra Sell, Stefan Busch, Dieter Rosskopf, Sebastian Virchow, Helmut Esche, Norbert Brockmeyer, Karl H. Jakobs, Winfried Siffert

the Institut fur Pharmakologie (F.P., A.M., A.S., S.B., D.R., S.V., K.H.J., W.S.), the Abteilung fur Kardiologie (M.M.), the Institut fur Molekularbiologie (H.E.), and the Hautklinik (N.B.), Universitatsklinikum Essen (Germany).

Correspondence to Dr Winfried Siffert, Institut fur Pharmakologie, Universitatsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have shown an enhanced signaling capacity of receptors coupled to pertussis toxin (PTX)–sensitive guanine nucleotide–binding proteins (G proteins) in immortalized B lymphoblasts from patients with essential hypertension. In the present study, we analyzed (1) whether such alterations would also be expressed in nontransformed cells of these individuals and (2) whether other G protein–mediated signaling pathways were also altered. Therefore, we established primary cultures of skin fibroblasts from previously characterized normotensive and hypertensive individuals (NT and HT cells, respectively). [Ca²⁺]_i rises induced by lyso-phosphatidic acid (LPA), thrombin, and sphingosine-1-phosphate as well as the formation of inositol 1,4,5-trisphosphate and [³H]thymidine incorporation evoked by LPA were PTX sensitive and enhanced twofold in HT fibroblasts. In contrast, cellular responses induced by bradykinin, endothelin-1, and angiotensin II (all PTX insensitive) were similar in NT and HT cells. Formation of cAMP induced by stimulation of G_s with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of G_i2, G_i3, Gß₂, and Gß₄. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits G_i2, G_i3, Gß₁, and Gß₂ from NT and HT cell lines yielded no evidence for mutations in these genes. Although the molecular mechanisms remain to be defined, these data support the concept of a selective enhancement of signal transduction via PTX-sensitive G proteins in essential hypertension.

Key Words: fibroblasts • lyso-phosphatidic acid • bradykinin • platelet-derived growth factor • proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Presently available data suggest that 30% to 50% of patients with EH display an enhanced Na⁺-H⁺ exchange activity in blood cells and that this phenotype coincides with an increased body mass index, left ventricular hypertrophy, insulin resistance, renal Na⁺ retention, low plasma renin, and, in the presence of type-1 diabetes, an increased susceptibility of developing nephropathy.^{1 2} Studies on Epstein-Barr virus–immortalized B lymphoblasts from normotensive and hypertensive subjects have shown that the phenotypes of "low" or "high" Na⁺-H⁺ exchange activity persist after prolonged cell culture, suggesting that these properties are under genetic control.³ In addition, HT cell lines with enhanced Na⁺-H⁺ exchanger activity proliferate distinctly faster, display enhanced DNA synthesis, and pass faster through the cell cycle than those from normotensive subjects with low Na⁺-H⁺ exchanger activity.^{3 4 5} It could be ruled out, however, that the enhanced Na⁺-H⁺ exchanger activity is caused by a mutation in the NHE-1 gene,³ by an overexpression of NHE-1 mRNA³ or protein,⁶ or by a differential posttranslational modification (eg, glycosylation) of the transporter.⁶

Recent studies revealed distinct differences in intracellular signal transduction between these NT and HT cell lines. HT lymphoblasts displayed enhanced [Ca²⁺]_i increases, IP₃ formation, and proliferation upon stimulation with platelet-activating factor and somatostatin.⁷ Interestingly, this difference in signal transduction was completely corrected for in cells treated with PTX, which blunts signal transduction via G_i-type guanine nucleotide–binding proteins (G proteins).⁷ Furthermore, by measuring agonist-stimulated binding of the stable GTP analogue GTPS to HT and NT cell lines, an enhanced receptor-induced activation of PTX-sensitive G proteins in HT lymphoblasts could be demonstrated directly.⁷ This enhanced activation of PTX-sensitive G proteins in HT cell lines was mimicked by mastoparan-7, a peptide mimetic of an activated G protein–coupled receptor. However, neither Western blot analysis nor ADP-ribosylation studies yielded any evidence for an overexpression of PTX substrates as the major reason for the enhanced G protein activation in HT lymphoblasts.⁷

Although these experiments have yielded novel insights into potential pathogenetic mechanisms that may finally result in EH in the group of patients with enhanced Na⁺-H⁺ exchanger activity, many questions remain to be answered. One of these relates to the issue of whether the enhanced G protein activation is restricted to immortalized B lymphoblasts, ie, cells that are not likely to play a major role in the pathogenetic mechanisms leading to EH, or whether this abnormality is expressed in nontransformed cells as well. Another limitation of the previously used B lymphoblast system resides in the fact that the repertoire of expressed G protein–coupled receptors is rather restricted. Thus, although our previous experiments demonstrated an enhanced signal transduction via PTX-sensitive G proteins in EH, an altered signaling by receptors coupled to other G protein subtypes could not be ruled out. To answer these questions, we have established primary cultures of skin fibroblasts from these previously characterized normotensive and hypertensive subjects. These cells offer the advantage of expressing a variety of different receptors that couple to different G proteins, thereby evoking distinct and well-characterized pathways of intracellular signal transduction. Furthermore, fibroblast receptor expression and intracellular effector systems display many similarities to those of cells of the cardiovascular system, eg, vascular smooth muscle cells, endothelial cells, and platelets, to name but a few. The data in the present study strongly suggest that enhanced G protein–mediated signal transduction in EH is restricted to those receptors that activate PTX-sensitive G proteins, whereas signaling by receptors coupled to PTX-insensitive G proteins, ie, G_s and G_q, is apparently unaltered. This enhanced responsiveness, however, is due to neither mutations in the genes encoding for the ubiquitously expressed PTX substrates G_i2 and G_i3 nor the ß subunits Gß₁ and Gß₂.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
LPA, bradykinin, thrombin, angiotensin II, ET-1, SPP, and PDGF were from Sigma Chemical Co, and [³H]thymidine (6.7 Ci/mmol) was from DuPont-New England Nuclear. The rabbit polyclonal antibodies directed against peptide epitopes on G_i/o/t/z, Gß₁, Gß₂, Gß₃, and Gß₄ were purchased from St. Cruz Biotechnology. The anti-G_i1/2 antibody was obtained from Calbiochem, and the peroxidase-conjugated goat anti-rabbit antibody was from Sigma. All other materials were from previously described sources.⁷

Individuals and Establishment of Fibroblast Cell Lines
Skin biopsies were made from previously characterized normotensive and hypertensive subjects,^{3 8} and some of the individuals' characteristics are displayed in the Table. In brief, hypertensive and normotensive subjects had been selected on the basis of high and low Na⁺-H⁺ exchange activity, respectively, in blood cells and a positive and negative family history of EH, respectively. Immortalized B lymphoblasts from the hypertensive subjects retained the enhanced Na⁺-H⁺ exchange activity after prolonged cell culture³ and, in addition, displayed enhanced signal transduction in terms of agonist-stimulated [Ca²⁺]_i signals, IP₃ formation, and GTPS binding.⁷ For the present study, we selected normotensive and hypertensive subjects who displayed lowest or highest agonist-evoked [Ca²⁺]_i signals in immortalized lymphoblasts. It should be noted that three of four hypertensive subjects were under antihypertensive therapy with either a ß-adrenoceptor blocker or a Ca²⁺ channel blocker. Although a definitive proof is lacking, we assume that potential drug effects on signal transduction in skin fibroblasts disappear in cells cultured for more than five passages.

View this table: [in this window] [in a new window]   Table 1. Characterization of Enrolled Individuals

The biopsy cylinders were cut into several thin slices and were cultured under a cover glass in six-well plates using DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere (95% O₂/5% CO₂, 37°C). When enough fibroblasts were grown out of the primary material, the cells were trypsinized (trypsin-EDTA, GIBCO-BRL) and transferred into small culture flasks (25-cm² culture area). Fibroblasts from passages 5 to 15 were used for all experiments. To avoid fortuitous influences of cell culture conditions, each of the different experiments was repeated within a time interval of >2 weeks.

[Ca²⁺]_i Measurements
Cells were grown to confluence and passaged at a ratio of 1:0.8 two days before the experiment. Thereafter, cells were growth-arrested in serum-free DMEM containing 100 U/mL penicillin and 100 µg/mL streptomycin for 20 to 24 hours. Cells were trypsinized, pelleted by centrifugation (10 minutes, 700g), resuspended in DMEM containing 1% (wt/vol) fatty acid–free BSA, and then incubated with 3 µmol/L fura 2-AM for 1 hour at 37°C. Thereafter, the cells were again pelleted, resuspended in DMEM, and stored at 37°C. Before each measurement, an aliquot (0.25 to 0.5x10⁶ cells per measurement) was transferred to Eppendorf tubes, centrifuged to remove extraneous dye, and resuspended in 2 mL prewarmed buffer containing (mmol/L) NaCl 135, KCl 5, MgCl₂ 1, CaCl₂ 1, D-glucose 10, and HEPES 20, pH 7.4. [Ca²⁺]_i measurements were performed on a spectrofluorometer (LS 5B, Perkin Elmer Corp) equipped with a thermostated cuvette holder and a fast filter application (ratio mode device) as described before.⁷

Quantification of IP₃ Formation
Fibroblasts were grown in six-well tissue culture dishes to subconfluent density and labeled with 5 µCi/mL myo-[³H]inositol in serum-free DMEM (without inositol) for 48 hours. Then, the cells were washed three times with phosphate-buffered saline and stimulated with the indicated agonists for 30 seconds at 37°C. Reactions were terminated by addition of 500 µL of 1.2 mol/L HCl and 3 mL of chloroform/methanol/HCl (200:100:0.75 [vol/vol/vol]) under vigorous shaking. After phase separation, the aqueous supernatants were collected, and the [³H]IP₃ formed was analyzed as described previously.⁷

Determination of cAMP Accumulation
Cells grown to subconfluence on 60-mm plates were incubated in serum-free DMEM for 30 minutes at 37°C under cell culture conditions (95% O₂/5% CO₂). Thereafter, the medium was changed to DMEM supplemented with 0.5 mmol/L 3-isobutyl 1-methylxanthine for 20 minutes, followed by cell stimulation with isoproterenol or forskolin for 5 minutes. Cellular cAMP levels were quantified using the method of Gilman.⁹

DNA Synthesis and Cell Proliferation
DNA synthesis was determined from [³H]thymidine incorporation. Fibroblasts were seeded onto 24-well plates at a density of 10 000 cells per well. After attachment of the cells overnight, cells were growth-arrested by incubation in DMEM supplemented with 0.5% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. After 48 hours, the cells were stimulated with different agonists. After another 24 hours, 0.5 µCi of [³H]thymidine was added to each well. Another 24 hours later, the medium was aspirated, and the cells were washed with phosphate-buffered saline and lysed with 200 µL of tissue solubilizer (Solvable NEF-910 G, DuPont). Radioactivity was measured by liquid scintillation counting after adding 4 mL of scintillation cocktail.

Immunoblotting
Crude fibroblast cell membranes were prepared by nitrogen cavitation as described previously.⁷ Since these nontransformed primary human skin fibroblasts proliferated very slowly, analysis of G protein subunit expression in single defined NT and HT fibroblast cell lines was impossible. Therefore, membranes from cell lines N1 to N4 and H1 to H4 were collected and pooled (see also "Discussion"). Protein concentration was determined according to Bradford,¹⁰ with bovine IgG used as a standard. Membrane proteins (25 µg) were heated for 5 minutes at 95°C in sample buffer containing 5% 2-mercaptoethanol. They were fractionated by SDS-PAGE according to Laemmli¹¹ with 10% ( subunits) or 12% (ß subunits) acrylamide in the running gel and 4% acrylamide in the stacking gel. Urea (6 mol/L) was present in part of the running gels to resolve _i2 from _i1 and _i3, respectively. Proteins were electrotransferred to nitrocellulose using a Trans-Blot electrophoretic transfer cell (Bio-Rad) at 100 V for 1 hour. Nitrocellulose filters were blocked for 1 hour at room temperature in TBS (10 mmol/L Tris-HCl [pH 8.0] and 150 mmol/L NaCl) containing 5% skimmed milk powder and 0.05% Tween 20. The filters were washed three times for 5 minutes each in TBS containing 0.05% Tween 20 (TBS/Tween 20) and subsequently incubated for 60 minutes at room temperature in the primary antibody, which was diluted to a final concentration of 0.1 µg/mL with TBS containing 1% BSA (TBS/BSA). The filters were washed four times for 5 minutes each in TBS/Tween 20 and incubated for 30 minutes at room temperature in the peroxidase-conjugated antibody, which was diluted at 1:5000 with TBS/BSA. The filters were washed three times for 5 minutes each in TBS/Tween 20 and once in TBS. Finally, the filters were treated with ECL reagent (Amersham) according to the manufacturer, and immunoreactive bands were visualized by autoradiography. Autoradiographs were scanned, and the density of the specific bands was quantified using the computer program Quantiscan (Biosoft)

Cloning and Sequencing of G Protein Subunits _i2, _i3, ß₁, and ß₂
RNA was prepared, and cDNAs were synthesized in oligo dT–primed RT reactions as described previously.³ From each of the two cell lines of NT and HT origin, cDNAs spanning the entire coding regions were obtained by RT-PCR. 5'-Oligonucleotide primers were as follows: GGCCGGGCCGGCGGACGGC (_i2), CGTGTCCCCTCTCCCGCCGC (_i3), GCATTGAAGAGCACTAAGATCG (ß₁), and CCCCCAACCCTGCCCCACG (ß₂). 3'-Oligonucleotide primers were as follows: TCCCGCCAGGCCCCGCTGC (_i2), CTTTCACTAACATCCATGCTTCTC (_i3), TGGCATCCACATGCTACTGGC (ß₁), and CCAGTGGGGGTGGGGCCA (ß₂). For each PCR product, 35 cycles consisting of 1 minute at 95°C for denaturation, 1 minute at 56°C for annealing, and 2 minutes at 72°C for extension were performed. In the last cycle, extension was for 9 minutes. After purification on agarose gels, the specific PCR products were directly ligated into a pGem5Z T-vector (Promega), and the ligation products were transformed into competent cells of the JM 109 strain. Clones containing cDNA inserts were selected by blue-white screening of ampicillin-resistant colonies. Plasmid DNA was prepared from those clones and selected for cDNA inserted in one specific direction by restriction analysis. For each cell line, mixtures of equal amounts of plasmid DNA from 50 clones were subjected to automated sequencing using fluorescent dyes. Sequences not unequivocally resolved in one direction were verified by sequencing of the opposite strand.

Presentation of Data and Statistics
Results are given as mean±SEM if not otherwise indicated. Data were analyzed using two-tailed Student's t test and regarded significantly different at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptor-Induced Increases in [Ca²⁺]_i and IP₃ Formation
First, we quantified LPA-, bradykinin-, and PDGF-induced [Ca²⁺]_i rises in each of the established four NT and four HT fibroblast cell lines. Mean values of basal [Ca²⁺]_i were calculated at 113±46 (n=208) and 112±36 (n=211) nmol/L, respectively, and were, thus, not significantly different. Upon stimulation by 100 nmol/L LPA, [Ca²⁺]_i rises were distinctly higher in HT fibroblasts compared with NT fibroblasts (Fig 1A), and changes in [Ca²⁺]_i averaged 560±75 versus 218±45 nmol/L (P<.01) in HT and NT fibroblasts, respectively. In contrast, [Ca²⁺]_i increased by 853±66 and 763±47 nmol/L in HT and NT fibroblasts, respectively, stimulated with 100 nmol/L bradykinin; these figures were not significantly different (P>.3, Fig 1B). LPA-evoked [Ca²⁺]_i signals were enhanced in HT fibroblasts at all concentrations studied and amounted to 234±45, 343±42, 560±75, and 561±34 nmol/L above baseline at 1, 10, 100, and 1000 nmol/L LPA, respectively (Fig 2A). The corresponding figures of NT fibroblasts averaged to 61±15, 165±31, 218±45, and 324±47 nmol/L above basal values upon stimulation with 1, 10, 100, and 1000 nmol/L LPA, respectively. Differences were statistically significant at all concentrations of LPA studied (P<.05 for NT versus HT). Upon treatment of fibroblasts with PTX (50 ng/mL, 5 hours), LPA (1 µmol/L)–evoked Ca²⁺ signals were strongly reduced and averaged 163±23 nmol/L (P<.02 for PTX versus control) and 199±34 nmol/L (P<.001 for PTX versus control) above baseline in NT and HT fibroblasts, respectively, and [Ca²⁺]_i rises were not significantly different (P>.4 for NT versus HT). This not only confirms that LPA stimulates receptors coupled to G_i-type G proteins¹² but also apparently supports our previous notion of a selectively enhanced signaling via PTX-sensitive G proteins in our highly selected hypertensive individuals.⁷ In contrast, bradykinin-evoked changes of [Ca²⁺]_i were similar in NT and HT fibroblasts at all concentrations applied (Fig 2B). These bradykinin-evoked [Ca²⁺]_i rises remained largely unaffected by PTX, and only a minor reduction by 10% to 15% was observed in some experiments (data not shown). This is in agreement with previous reports demonstrating that this peptide activates cellular signaling mechanisms via the PTX-insensitive G_q protein.¹³ Interestingly, rises in [Ca²⁺]_i were also enhanced in HT fibroblasts stimulated by 25 ng/mL PDGF (Fig 2C). The average increases in [Ca²⁺]_i evoked by this growth factor amounted to 317±31 and 210±31 nmol/L in HT and NT fibroblasts, respectively (P<.05). Surprisingly, PDGF-induced [Ca²⁺]_i increases were also partially PTX sensitive, with the observed inhibition ranging from 30% to 50% (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 1. LPA- and bradykinin-induced rises in [Ca2+]i in individual NT and HT fibroblast cell lines. Displayed are [Ca2+]i rises in NT (open symbols) and HT (filled symbols) cell lines upon stimulation with 100 nmol/L LPA (A) or 100 nmol/L bradykinin (B). Each symbol represents the mean of three measurements performed on a given day. The number of symbols indicates the number of independent determinations (each in triplicate), which were conducted at >2-week intervals. The different cell lines are designated N1 to N4 (NT cell lines) and H1 to H4 (HT cell lines), and the respective individuals refer to those characterized previously7 and in the Table. Dashed lines indicate means of all NT and HT fibroblast cell lines.

View larger version (20K): [in this window] [in a new window]   Figure 2. LPA-, bradykinin-, and PDGF-evoked [Ca2+]i signals in NT and HT fibroblasts. Skin fibroblasts from normotensive (open columns) and hypertensive (filled columns) subjects were stimulated with LPA (1 to 1000 nmol/L, A), bradykinin (1 to 100 nmol/L, B), or PDGF (20 ng/mL, C). In some experiments, cells were pretreated with 50 ng/mL PTX for 4 hours and subsequently stimulated with 1 µmol/L LPA (A). Each column represents values from four NT and HT cell lines each, in which [Ca2+]i determinations have been conducted in triplicate in at least two independent measurement series, each constituting two to three determinations for each agonist concentration. Values are mean±SE. *P<.05 vs NT.

To further substantiate our hypothesis of an enhanced G_i -type G protein activation in the established HT fibroblast cell lines, we quantified [Ca²⁺]_i rises upon stimulation with four additional different agonists that are known to activate intracellular signal transduction pathways via PTX-sensitive and/or PTX-insensitive G proteins. The putative receptor for SPP has been proposed to predominantly activate PTX-sensitive G proteins in a variety of cell types.¹⁴ SPP (1 µmol/L) increased [Ca²⁺]_i by 227±44 and 403±19 nmol/L above baseline in NT and HT fibroblasts (Fig 3A), respectively (P=.013). In PTX-treated cells, this difference was completely abrogated (Fig 3A), and [Ca²⁺]_i rises averaged 115±9 versus 125±21 nmol/L in NT and HT fibroblasts (P>.4 for NT versus HT), respectively. This effect of PTX on SPP-induced Ca²⁺ signals was statistically significant (P<.05 for control versus PTX). Thrombin, which activates PTX-sensitive as well as PTX-insensitive G proteins,¹⁵ also induced enlarged Ca²⁺ signals in HT fibroblasts (Fig 3B). [Ca²⁺]_i rises evoked by thrombin (10 U/mL) averaged 46±6 versus 121±17 nmol/L above baseline (P<.025) in NT and HT fibroblasts, respectively. Again, this difference was almost completely abrogated by PTX (Fig 3B). Whereas PTX reduced the thrombin-evoked [Ca²⁺]_i rise only slightly in NT fibroblasts (to 30±1 nmol/L, P<.05 versus control), its effect was much more marked in HT fibroblasts, with mean [Ca²⁺]_i rises being calculated at 41±2 nmol/L (P<.01 versus control). Angiotensin II–mediated signal transduction via the AT₁ receptor can involve PTX-sensitive and/or PTX-insensitive G proteins, depending on cell type or tissue investigated.^{16 17} Angiotensin II–evoked Ca²⁺ signals were slightly, albeit not significantly, higher in HT compared with NT cell lines (201±33 versus 141±19 nmol/L, P=.14, Fig 3C). PTX did not significantly reduce the angiotensin II–induced [Ca²⁺]_i rise in NT fibroblasts ([Ca²⁺]_i=121±23 nmol/L, P>.5 versus control). In HT fibroblasts, the PTX sensitivity of angiotensin II–induced Ca²⁺ signals was somewhat more pronounced ([Ca²⁺]_i=142±23 nmol/L) but still not significantly different from untreated controls (P>.2). Thus, angiotensin II–evoked Ca²⁺ signals appear to be predominantly mediated by PTX-insensitive G proteins in primary human skin fibroblasts and were, in agreement with the concept proposed here, not significantly different in NT and HT fibroblasts. Finally, we stimulated NT and HT fibroblasts with ET-1. ET-1–evoked Ca²⁺ signals showed a tendency to be slightly higher in HT compared with NT fibroblasts (178±21 versus 133±19 nmol/L, P=.16). This tendency disappeared after PTX treatment ([Ca²⁺]_i=106±14 and 102±26 nmol/L for NT and HT fibroblasts, respectively). Identification of the endothelin receptor subtype mediating the ET-1–induced Ca²⁺ signals in human skin fibroblasts was beyond the scope of the present study.

View larger version (27K): [in this window] [in a new window]   Figure 3. SPP–, thrombin (Thr)–, angiotensin II (Ang II)–, and ET-1–evoked [Ca2+]i signals in NT and HT fibroblasts. Skin fibroblasts from normotensive (open columns) and hypertensive (filled columns) subjects were stimulated with SPP (1 µmol/L, A), Thr (10 U/mL, B), Ang II (1 µmol/L, C), or ET-1 (1 µmol/L, D). In some experiments, cells were pretreated with 50 ng/mL PTX for 4 hours and subsequently stimulated with agonists, as indicated. Each column represents values from four NT and HT cell lines each, in which [Ca2+]i determinations have been conducted in triplicate in at least two independent measurement series. Values are mean±SE. *P<.05 vs NT.

Subsequently, we quantified the formation of IP₃ in NT and HT fibroblasts stimulated with 100 nmol/L LPA or 100 nmol/L bradykinin (Fig 4). LPA-induced IP₃ formation, which was completely blunted in cells pretreated with PTX, was significantly higher in HT than in NT fibroblasts and averaged 324±50% versus 219±36% of unstimulated controls (P<.05). In contrast, PTX-insensitive bradykinin-induced IP₃ formation was not significantly different in HT and NT fibroblasts.

View larger version (18K): [in this window] [in a new window]   Figure 4. LPA- and bradykinin-induced IP3 formation in NT and HT fibroblasts. NT (open columns) and HT (filled columns) fibroblasts labeled with [3H]inositol and pretreated or not with 100 ng/mL PTX were stimulated with 100 nmol/L LPA or 100 nmol/L bradykinin as indicated. [3H]IP3 formation was quantified as detailed in "Experimental Procedures." IP3 formation is expressed as percentage of controls, the 100% value reflecting IP3 levels in unstimulated fibroblasts. Basal IP3 levels were not different in NT and HT fibroblasts. Values are mean±SD from each of the four NT and HT cell lines. *P<.05 vs NT.

cAMP Formation in Fibroblasts
To study whether receptor signaling via G_s, the adenylyl cyclase–stimulatory G protein, is altered, NT and HT fibroblasts were stimulated with isoproterenol, an agonist of ß-adrenoceptors, coupled via G_s to adenylyl cyclase (Fig 5). Upon stimulation with 10 µmol/L isoproterenol, cAMP increased by factors of 6.1±1.2 and 4.3±2.2 above baseline in NT and HT fibroblasts, respectively (P>.2). When NT and HT fibroblasts were stimulated with 50 µmol/L forskolin, which directly activates adenylyl cyclase, cAMP formation was also similar in NT and HT fibroblasts and averaged to 16.2±7.3- and 17.6±9.9-fold above basal values, respectively. Thus, signal transduction via G_s is apparently not enhanced in HT fibroblasts. Subsequently, we quantified the inhibition by LPA of forskolin-stimulated cAMP formation in NT and HT fibroblasts. In these experiments, cells were stimulated with 50 µmol/L forskolin in the presence of 10 µmol/L LPA. LPA-induced inhibition of forskolin-stimulated cAMP formation was fully PTX sensitive (data not shown) and almost identical in NT and HT fibroblasts (Fig 5). Thus, cAMP formation averaged to 5.1±1.9- and 4.3±1.0-fold above basal values in NT and HT fibroblasts stimulated with 50 µmol/L forskolin in the presence of LPA.

View larger version (16K): [in this window] [in a new window]   Figure 5. Adenylyl cyclase activation in NT and HT fibroblasts. NT (open columns) and HT (filled columns) fibroblast cell lines were stimulated with 10 µmol/L isoproterenol, 50 µmol/L forskolin, or 50 µmol/L forskolin plus 10 µmol/L LPA, as indicated. cAMP formation was quantified as detailed in "Experimental Procedures" and is given as x-fold above control, ie, cAMP levels in unstimulated cells. Columns represent mean±SD from each of the four NT and HT cell lines, in which triplicate determinations had been performed.

Receptor-Regulated DNA Synthesis
DNA synthesis was quantified in NT and HT fibroblasts stimulated with LPA (10 µmol/L), PDGF (20 ng/mL), or FCS (10%) by measuring the incorporation of [³H]thymidine. DNA synthesis in response to LPA was completely PTX sensitive, whereas that induced by PDGF and FCS was reduced by 55% and 75%, respectively (Fig 6A). Bradykinin did not increase DNA synthesis. In NT fibroblasts (Fig 6B), LPA, PDGF, and FCS increased [³H]thymidine incorporation to 152±7%, 150±15%, and 214±22% of unstimulated controls, respectively (mean±SE). The corresponding values in HT fibroblasts were 196±16%, 278±22%, and 435±82%, respectively. Thus, DNA synthesis initiated via fully (LPA) or partially (PDGF, FCS) PTX-sensitive signal transduction pathways was significantly enhanced in HT fibroblasts.

View larger version (25K): [in this window] [in a new window]   Figure 6. DNA synthesis in NT and HT fibroblasts. DNA synthesis was determined by measuring the incorporation of [3H]thymidine in cells stimulated with 10 µmol/L LPA, 10 ng/mL PDGF, or 10% FCS. A, Effect of PTX (100 ng/mL) on [3H]thymidine incorporation evoked by these stimuli. Data represent mean±SD and were obtained from four NT and 2 HT cell lines. B, [3H]thymidine incorporation into four NT (open columns) and four HT (filled columns) cell lines (mean±SD, triplicate determinations each). Control (100% value) is defined as [3H]thymidine incorporation into fibroblasts kept in 0.5% FCS. *P<.05 vs NT.

Expression of G Protein and ß Subunits
To investigate whether HT fibroblasts would potentially overexpress PTX-sensitive G proteins, we performed Western blot analysis using various antibodies directed against different G protein subunits. In Fig 7A (left blot) is displayed a representative analysis of -subunit expression as assessed using an anti-G_i,common antibody. This antibody detected two discrete bands of similar density in NT and HT fibroblast cell lines as well as in bovine brain membranes. An anti-G_i1,2 antibody, on the other hand, detected only one single band in NT and HT fibroblasts, respectively, but two discrete bands in brain membranes (Fig 7A, right blot). Thus, human skin fibroblasts predominantly express G_i2 and G_i3, but not G_i1, which was also confirmed by RT-PCR analysis (see below). Densitometric evaluation of these bands yielded no evidence for different amounts of G_i2/3 subunits in NT and HT fibroblasts. The anti-Gß₁ antibody detected only very faint bands in membranes from NT and HT fibroblasts (not shown), although this subunit could be easily detected and cloned by RT-PCR (see below). Strong bands corresponding to molecular weights of 37 kD were detected by specific anti-Gß₂ and anti-Gß₄ antibodies (Fig 7B), which were again not significantly different in NT and HT fibroblasts. The anti-Gß₃ antibody, on the other hand, detected multiple bands (not shown), all of which appeared unspecific. To determine which potential differences in G protein -subunit expression are detectable with our assay system, we blotted different amounts of membrane protein and probed the blots with an anti-_i,common antibody. In the representative result displayed in Fig 7C, relative densities of the stained bands were determined at <10, 65, 132, 952, and 3828 arbitrary units for 10, 20, 30, 50, and 75 µg of protein. We conclude from these data that a 50% to 70% difference in the amount of G proteins expressed in NT and HT cells would not have escaped detection.

View larger version (48K): [in this window] [in a new window]   Figure 7. Western blot analysis of G protein subunit expression in NT and HT fibroblasts. Membranes from cell lines N1 to N4 and H1 to H4 were pooled separately, and 20 µg of protein was transferred to nitrocellulose as described in "Experimental Procedures." Membranes from bovine brain served as controls as indicated. Blots were probed with an anti-Gi,common antibody (A, left blot), with an antibody directed against Gi1,2 (A, right blot), or with antibodies against Gß2 (B, left blot) or Gß4 (B, right blot). The positions of standard molecular weight markers are indicated. Shown are results from each one representative experiment. C, Representative experiment in which different amounts of membrane protein were blotted as indicated, followed by probing of blots with an anti-Gi,common antibody. It is concluded that a 50% to 70% difference in G protein expression would not have escaped detection in our assays.

Cloning and Sequencing of G_i2, G_i3, Gß₁, and Gß₂
Since the data accumulated so far indicated a selectively enhanced signaling via PTX-sensitive G proteins in HT cells (lymphoblasts and fibroblasts), without any indications for an overexpression of G proteins, we searched for potential mutations in genes encoding for and ß subunits of PTX-sensitive G proteins. G_i2, G_i3, Gß_1, and Gß₂ were considered as potential candidates, since these subunits are ubiquitously expressed,¹⁸ including in human lymphoblasts and fibroblasts as confirmed by RT-PCR analysis (not shown). In contrast, the expression of the PTX-sensitive G proteins G_o and G_i1 is far more restricted.¹⁸ Nucleotide sequences of G_i2, G_i3, Gß_1, and Gß₂ were identical in NT and HT cells, excluding a mutation in these proteins as the primary reason for the enhanced signal transduction.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies on immortalized B lymphoblasts from patients with EH and enhanced Na⁺-H⁺ exchanger activity have revealed an enhanced activation of PTX-sensitive G proteins.⁷ This conclusion was mainly based on two findings. First, HT lymphoblasts displayed enhanced [Ca²⁺]_i signals upon stimulation with platelet-activating factor and somatostatin. Pretreatment with PTX strongly reduced these agonist-evoked Ca²⁺ signals, and the residual Ca²⁺ responses were no longer different between NT and HT cell lines. Second, both receptor-mediated stimulation and direct (by mastoparan-7) stimulation of GTPS binding to PTX-sensitive G proteins were significantly increased in HT lymphoblasts.⁷ Unfortunately, B lymphoblasts apparently do not express functional receptors that are selectively coupled to PTX-insensitive G proteins, eg, G_q or G_s. Therefore, our proposal of a selective enhancement of signal transduction via PTX-sensitive G proteins in HT cell lines was based on circumstantial evidence but could not be confirmed experimentally. Thus, one of the major reasons that we established primary cultures of skin fibroblasts from the previously characterized normotensive and hypertensive individuals was because these cells express a variety of receptors that activate fairly well-described signal transduction mechanisms involving either G_i, G_q, or G_s. Clearly, the present study was not intended to be an epidemiological one but aimed at further characterizing potential changes in intracellular signal transduction pathways in previously characterized, highly selected normotensive and hypertensive individuals (see Table and Reference 7). Therefore, and because the experiments presented here were conducted on fibroblasts from only four hypertensive individuals, only speculations are possible regarding the prevalence of the "enhanced G protein activation phenotype" in the overall hypertensive population. Our patients had originally been selected on the basis of elevated Na⁺-H⁺ exchanger activity in blood cells.⁸ Available evidence suggests that this abnormality can be found in 30% to 50% of patients with EH.^{1 19} If the reason for enhanced Na⁺-H⁺ exchanger activity were the same in these patients, ie, increased responsiveness of PTX-sensitive G proteins, it could be estimated that also 30% to 50% of patients with EH may display the characteristics described here.

During the work with the established fibroblast cell lines, two major disadvantages of this system became apparent. In contrast to immortalized cells, primary cultures of skin fibroblasts proliferate very slowly, which made it very difficult to collect sufficient cell material for the repetitive experiments reported in the present study. This difficulty made it impossible, for example, to determine agonist-induced GTPS binding as a direct measure of G protein activation or dose-response curves for all agonist-mediated cellular responses reported here, since the amount of cell material required for such experiments distinctly exceeded its availability. Second, the senescence of these cells, which became apparent at passages >20, made it imperative that all experiments were conducted at passages between 5 and 15, which imposed further limits on the number and diversity of possible experiments. Despite these limitations, the findings reported here provide novel insights into potential pathogenetic mechanisms in EH that significantly exceed those reported previously.

The results reported herein demonstrate that LPA-, SPP-, and thrombin-induced cellular responses, eg, [Ca²⁺]_i increase, all of which were strongly PTX sensitive, were significantly enhanced in HT fibroblasts, with the exception of adenylyl cyclase inhibition (see below). The fact that this enhancement was seen with three different chemically unrelated agonists makes it unlikely, in our view, that increased agonist affinity of receptors in HT fibroblasts could explain the enhanced cellular reactivity. In contrast, PTX-insensitive signaling via receptors for bradykinin, angiotensin II, and ET-1, which is presumably mediated by G_q-type G proteins, was similar in NT and HT fibroblasts. Furthermore, isoprenaline-induced formation of cAMP, which involves the adenylyl cyclase–stimulatory G protein, G_s, was almost identical in NT and HT fibroblasts. On the other hand, the observation of a virtually identical inhibition by LPA of forskolin-stimulated cAMP formation, although PTX sensitive, was somewhat surprising. Several facts may account for this apparent inconsistency with the general hypothesis proposed here. First, we had to apply relatively high concentrations of LPA (10 µmol/L) to induce a substantial and well-reproducible inhibition of the adenylyl cyclase. It appears possible that differences in LPA-induced adenylyl cyclase inhibition between NT and HT fibroblasts would have been observed at lower agonist concentrations. Unfortunately, the establishment of complete dose-response curves was not feasible because of the lack of sufficient cell material (see above). In addition, the existence of different isoforms of adenylyl cyclase has to be considered, and these isoforms differ with respect to activation and inhibition by G protein and ß subunits.²⁰ It remains unknown which of the hitherto cloned adenylyl cyclase isoforms is expressed in human skin fibroblasts. Finally, it has to be considered that the potency of G protein ß subunits to activate or inhibit adenylyl cyclases is 10-fold higher than that necessary to activate phospholipase Cß.²¹ It appears possible, therefore, that reduced activation of G protein subunits in NT fibroblast still releases sufficient ß subunits for substantial inhibition of adenylyl cyclase but that their availability becomes limiting with regard to phospholipase Cß activation. These issues require clarification in future experiments.

In general, the findings reported in the present study support our previous suggestion that signal transduction in which PTX-sensitive G proteins are involved is enhanced in EH. Pretreatment of the fibroblasts with PTX reduced PDGF-induced [Ca²⁺]_i increase and DNA synthesis by up to 50%, and in agreement with the enhanced activation of PTX-sensitive G proteins, PDGF-induced enhanced [Ca²⁺]_i rises and DNA synthesis were significantly enhanced in HT fibroblasts. The PTX sensitivity of PDGF signaling may appear surprising at first glance. PDGF belongs to the group of growth factors that are believed to activate signal transduction pathways involving receptor tyrosine kinases without the contribution of G proteins (for review see Reference 22). Nevertheless, evidence has been accumulated that growth factor signaling, at least in some cell types, may also involve G protein activation. For example, activation of hepatocytes by epidermal growth factor has been shown to be partially PTX sensitive.^{23 24} Furthermore, activation of the mitogen-activated protein kinase by the insulin-like growth factor 1 receptor in Rat-1 fibroblasts was recently shown to involve ß subunits released from G_i-type G proteins,²⁵ and finally, the PDGF-stimulated growth of aortic smooth muscle cells from guinea pig was found to be strongly inhibited by PTX.²⁶ Taken together, signaling via growth factor receptors with intrinsic tyrosine kinase activity could well account for G protein activation.^{27 28} Whether such a mechanism actually underlies the findings reported here remains unresolved. It appears also possible that PDGF stimulation of fibroblasts induces the release of compounds, eg, sphingosine derivatives,²⁹ which in turn stimulate PTX-sensitive G proteins,²⁹ thereby initiating [Ca²⁺]_i rises and DNA synthesis. However, further elucidating these mechanisms was clearly beyond the scope of the present study.

Besides providing experimental evidence that PTX-sensitive signaling pathways are enhanced in these HT cell lines, two additional major conclusions can be drawn from the present study. First, the fact that this enhanced signal transduction is also present in nontransformed primary cell lines rules out the possibility that this abnormality arises as an artifact of immortalization, with HT and NT lymphoblasts being differentially influenced by the Epstein-Barr virus genome. Second, the expression of an enhanced signal transduction in both (B lymphoblasts and fibroblasts) of these selected hypertensive individuals lends more credit to the notion that this abnormality may be inherited and potentially expressed in other body cells as well. Thus, we hypothesize that similar alterations are expressed in vascular smooth muscle cells, platelets, endothelial cells, and cardiomyocytes of these individuals, which then display an enhanced cellular response if stimulated by an agonist activating PTX-sensitive G protein–coupled receptors.

The major question to be answered in the future relates to the molecular reason(s) for this enhanced G protein activation. Heterotrimeric G proteins consist of and ß subunits, which display a marked diversity in terms of cloned isoforms and selective tissue distribution. Presently available evidence suggests that only the _i2 and _i3 isoforms of PTX-sensitive G proteins are expressed in lymphoblasts and fibroblasts, whereas the expression pattern of _i1 and the two _o isoforms is rather restricted. Earlier studies have made an overexpression of PTX substrates in HT cells as the reason for enhanced signal transduction unlikely.⁷ The results presented here add further support to this previous assumption, since from Western blot analysis of NT and HT fibroblasts, no indication for an overexpression of G_i2, G_i3, Gß₂, or Gß₄ was evident. This finding is in full agreement with those obtained in human platelets.³⁰ The apparent lack of expression of Gß₃ in fibroblasts is consistent with findings by Hansen et al,³¹ whereas the weak expression of Gß₁ protein was unexpected.

We could rule out mutations in the genes encoding for G_i2, G_i3, Gß₁, or Gß₂ to be responsible for the reported alterations. However, altered posttranslational modification(s) of these subunits, eg, lipid modifications,³² which could be functionally important, have not yet been excluded. Gß₄, which from Western blot analysis appears to be abundantly expressed in skin fibroblasts, has not yet been sequenced, because only a mouse sequence is available for the encoding cDNA,³³ and Gß₅ appears brain specific.³⁴ It must also be emphasized that the chosen experimental approaches are not likely suitable for detecting potential splice variants of the above G protein subunits, but experiments addressing this issue are currently being performed.

Not only subunits but also the ß dimers play a decisive role in the efficacy of receptor–G protein coupling. ß subunits can combine with different subunits.³⁵ Among these, G₅, G₇, G₁₀, G₁₁, and G₁₂ appear expressed in most tissues,^{35 36 37 38} whereas the expression of G₁, G₂, G₃, and G₈ may be tissue specific.^{31 36 37 39} Alterations in primary structure or posttranslational processing of any of these subunits cannot yet be excluded.

Although the molecular reason underlying the enhanced G protein activation in HT cells remains to be elucidated, the findings in the present study may significantly contribute to the clarification of some unresolved issues and put forward a hypothesis regarding the pathogenesis of EH in that group of patients that we are investigating. One major issue concerns conflicting results regarding the potential cellular overreactivity in EH. Platelets have often been used as a model to study cellular signal transduction in hypertension, but the results reported from different groups were contradictory. For example, thrombin has been observed to evoke increased [Ca²⁺]_i rises in platelets from hypertensive patients,^{40 41} whereas others could not find such increased [Ca²⁺]_i signals upon stimulation of platelets with vasopressin.^{42 43} This discrepancy may be explained in terms of differential G protein activation by these agonists. The platelet V₁ vasopressin receptor appears to predominantly activate G_q-type G proteins,⁴⁴ the activation of which appears unchanged in EH (see above). In contrast, PTX-sensitive G proteins, which are apparently readily activated in EH, significantly contribute to thrombin-induced platelet activation.⁴⁵

On the basis of the findings presented here, we would like to propose a hypothetical scenario to explain pathogenetic mechanisms that could be operative in those patients with essential hypertension who display the enhanced Na⁺-H⁺ exchanger phenotype. We propose that the basic abnormality resides in an enhanced activation of PTX-sensitive G proteins. This would explain increased immediate cellular responses, such as platelet activation and vasoconstriction, provided that these cells are stimulated with an appropriate agonist. Since G_i-type G proteins are also important mediators of cell proliferation, we propose that the described abnormality could also significantly contribute to abnormal growth phenomena in EH, eg, left ventricular hypertrophy (which, in some individuals, may develop before the establishment of hypertension⁴⁶ ) and/or media hypertrophy of blood vessels. Potentially, the phenotype described here could also constitute the missing link between hypertension, atherosclerosis, and coronary heart disease. Available concepts propose an important role for cytokines, such as PDGF, in the development of these disorders.^{47 48} Provided that signal transduction of PDGF partially involves PTX-sensitive G proteins not only in skin fibroblasts but also in vascular smooth muscle cells and cardiac fibroblasts, enhanced signaling via such G proteins could be a major step in the development of these hypertension-associated complications. Finally, the proposed "G protein concept of hypertension" appears ideally suited to fit within the framework of current understandings regarding the contribution of genes versus environment in the pathogenesis of hypertension. Thus, an enhanced activation of PTX-sensitive G proteins would provide the inherited susceptibility for hypertension without necessarily being sufficient to establish hypertension by itself. This proposed genetic background may become important only when the respective signal transduction pathways are excessively stimulated. One can easily envisage exogenous factors, eg, smoking or stress, which by increasing circulating hormones/neurotransmitters could increase the frequency by which this enhanced signal transduction mechanism is in use, which then could be a decisive or aggravating step in the development of hypertension and related disorders.

	Selected Abbreviations and Acronyms

 

EH = essential hypertension ET-1 = endothelin-1 GTPS = guanosine 5'-[-thio]triphosphate HT, NT cells = cell lines established from hypertensive and normotensive subjects IP3 = inositol 1,4,5-tris-phosphate LPA = lyso-phosphatidic acid PCR = polymerase chain reaction PDGF = platelet-derived growth factor PTX = pertussis toxin RT = reverse transcriptase SPP = sphingosine-1-phosphate TBS = Tris-buffered saline

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft, the Deutsche Stiftung fur Herzforschung e.V. (Frankfurt/Main), and the Fritz Thyssen-Stiftung. Dr Pietruck is the recipient of a fellowship from the Deutsche Forschungsgemeinschaft. The technical assistance of Gerlinde Siffert (Institut fur Pharmakologie) and Ursula Schmucker (Institut fur Molekularbiologie) is greatly appreciated.

Received April 15, 1996; accepted August 26, 1996.

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