G protein beta3 subunit 825T allele and hypertension.
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Alleles↗
Biomedical subjects
W Siffert
Publications and source records attributed to W Siffert.
Worldwide ethnic distribution of the G protein beta3 subunit 825T allele and its association with obesity in Caucasian, Chinese, and Black African individuals.
Recently, it was demonstrated that one allele (825T) of the gene encoding the G protein beta3 subunit (GNB3) is associated with hypertension in Germans. This study investigates a possible association with obesity in young male Germans, Chinese, and black South Africans with low, intermediate, and high 825T allele frequencies, respectively. In each of these three distinct cohorts, the 825T allele frequency was increased significantly in overweight (body mass index [BMI] > or =25 kg/m2) and obese individuals (BMI >27 kg/m2) compared to those with normal weight. The 825T allele frequencies in these three BMI groups were, respectively, 29.5, 39.3, and 47.7% in Germans, 46.8, 53.9, and 58.6% in Chinese, and 83.1, 87.7, and 90.9% in South Africans. In each of these three distinct groups, the 825T allele was significantly associated with obesity with odds ratios between 2 and 3. More urban than rural black Africans were overweight despite similar 825T allele frequencies in both populations, which underscores the role of both genetic and environmental factors. BP values in young male whites increased significantly with increasing BMI values but were independent of the C825T polymorphism, suggesting that hypertension associated with the 825T allele could be a consequence of obesity. Genotyping of 5254 individuals from 55 native population samples from Africa, the Americas, Europe, Asia, Australia, and New Guinea demonstrated highest 825T allele frequencies in black Africans (82%) and intermediate values in east Asians (47%). It is anticipated that high frequencies of the 825T allele in Africans and Asians may contribute to an obesity and hypertension epidemic if Westernization of lifestyles continues.
Adolescent↗
Enhanced fMLP-stimulated chemotaxis in human neutrophils from individuals carrying the G protein beta3 subunit 825 T-allele.
We have recently described a C825T polymorphism in the gene encoding for the Gbeta3 subunit of heterotrimeric G proteins. The 825T allele is associated with a novel splice variant (Gbeta3-s) and enhanced signal transduction via pertussis toxin (PTX)-sensitive G proteins. fMLP-induced chemotaxis, but not O2- generation, was increased in neutrophils with the TC/TT (EC50 = 1.5 +/- 1.3 nM) genotypes compared to the CC genotype (EC50 = 5.9 +/- 1.5 nM). Maximal fMLP-induced increase in [Ca2+]i was significantly reduced in neutrophils from individuals with TC/TT genotype vs. CC genotype (212.9 +/- 10.1 nM vs. 146.4 +/- 24.2 nM). Gbeta3-s appears to be associated with enhanced immune cell function in humans.
Alleles↗
Enhanced G protein activation in IDDM patients with diabetic nephropathy.
Genetic susceptibility contributes significantly to the risk of developing nephropathy in insulin-dependent diabetes mellitus (IDDM). The cellular substrate for this has remained enigmatic. We investigated whether afflicted IDDM patients display an enhanced activation of pertussis toxin (PTX)-sensitive G proteins, a phenomenon which has been demonstrated in patients with essential hypertension. We established immortalised B lymphoblast cell lines from 10 IDDM patients without nephropathy (DC) and 15 IDDM patients with nephropathy (DN). Nephropathy was defined as a persistent albumin excretion rate of more than 20 microg/min (DC 3.9 +/- 5.8, DN 562.3 +/- 539.0 microg/min, respectively). Subjects were matched with regard to age (DC 28.9 +/- 6.5, DN 35.9 +/- 9.9 years), diabetes duration (DC 19.3 +/- 6.9, DN 22.7 +/- 5.8 years) and HbA1c values (DC 8.5 +/- 1.4, DN 8.8 +/- 1.6%). Reactivity of PTX-sensitive G proteins was quantified by measuring platelet-activating factor (PAF)-induced Ca2+ mobilisation (fura 2 method) and by mastoparan-stimulated [35S]GTPgammaS binding. Expression of Galphai proteins was quantified by Western blot analysis. PAF-evoked Ca2+ increases above baseline averaged 77.0 +/- 52.5 nmol/l in DC and 150.7 +/- 61.5 nmol/l in DN (p = 0.005). PAF-evoked Ca2+ increases correlated with stimulated [35S]GTPgammaS binding (r2 = 0.42, p = 0.012). From Western blot analysis an overexpression of Galphai proteins could be excluded in DN. A consequence of the altered metabolic milieu in diabetes is the increased release of vasoactive and proliferative agonists which promote glomerular hyperfiltration, hypertrophy, enhanced matrix deposition, and, finally, glomerulosclerosis. Many of these auto- and paracrine agonists bind to G protein-coupled receptors. Therefore, their cellular effects are reinforced by the enhanced G protein reactivity and increase the propensity to nephropathy in IDDM.
Adult↗
Expression, functional characterization and tissue distribution of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE).
We examined the functional properties of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE) upon stable transfection into PS120 fibroblasts which lack endogenous Na+/H+ exchange. In contrast to untransfected cells, XL-NHE-transfected cells displayed Na+-dependent alkalinization upon acidification with nigericin. XL-NHE activity was inhibited by amiloride, ethylisopropylamiloride, HOE694 [(3-methylsulphonyl-4-piperidinobenzoyl)-guanidine methanesulphonate] and HOE642 [4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulphonate], Ki values being calculated at 5 micromol/l, 25 nmol/l, 300 nmol/l and 180 nmol/l, respectively. The Na+ dependence of pHi recovery was compatible with simple Michaelis-Menten kinetics, the Km for Na+ being 22.0+/-3.2 mmol/l and the Hill coefficient for Na+ being approximately 1. XL-NHE was activated by phorbol ester, whereas forskolin exerted no effect, suggesting the involvement of phospholipase C/protein kinase C signalling pathways rather than protein kinase A signalling pathways in XL-NHE stimulation. Using reverse transcription polymerase chain reaction, XL-NHE message could be detected in various Xenopus tissues including heart, brain, skeletal muscle, reticulocytes, A6-kidney cells and oocytes.
Amiloride↗
Association of a human G-protein beta3 subunit variant with hypertension.
Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.
Alleles↗
G proteins and hypertension: an alternative candidate gene approach.
Hypertension affects approximately 20 to 30% of individuals in industrialized countries, and is commonly believed to develop on the basis of both genetic and environmental factors. The identification of genes susceptible to the most frequent form of hypertension, commonly referred to as "essential" hypertension, is hampered by the fact that blood pressure is a poorly defined phenotype that is modulated by multiple factors, such as gender, race, body mass etc., and that the definition of hypertension depends on a rather arbitrarily chosen cut-off value. Hence, more progress has been made in the identification of genes responsible for rare autosomal dominant forms of hypertension, such as Liddle's disease. This review focuses on an experimental approach that attempts to define candidate genes for essential hypertension using immortalized cells from well characterized normotensive and hypertensive subjects. From the presently available results, one attractive speculation is that an increased intracellular signal transduction caused by an enhanced reactivity of Gj-type G proteins represents a genetically fixed trait that renders affected individuals susceptible to essential hypertension.
B-Lymphocytes↗
Growth factor-like action of lysophosphatidic acid on human B lymphoblasts.
Proliferation and immunoglobulin secretion of B lymphocytes are regulated by specific antigens and numerous accessory immunomodulatory factors. Lysophosphatidic acid (LPA) is a glycerophospholipid mediator that is released from activated blood platelets, attains high levels in serum, and exerts potent stimulatory effects on, e.g., neutrophils, monocytes, and T lymphocytes. LPA is also generated by a secretory, cytokine-inducible phospholipase A2 present in high concentrations in inflammatory exudates and septic states. We investigated effects of LPA on human Epstein-Barr virus-immortalized B lymphoblasts, a model for immunoglobulin-secreting B cells. Intracellular Ca2+ was determined with fura 2 and the formation of inositol 1,4, 5-trisphosphate by anion-exchange chromatography. LPA stimulated an increase in inositol 1,4,5-trisphosphate levels and induced a transient rise in intracellular free Ca2+ concentration from 105 +/- 17 to 226 +/- 21 nM. This Ca2+ signal resulted from Ca2+ mobilization and Ca2+ influx and was subject to homologous desensitization. Pertussis toxin inhibited these responses by approximately 70%. Furthermore, LPA stimulated a 27.5% increase in guanosine 5'-O-(3-thiotriphosphate) binding to permeabilized B lymphoblasts, which suggests the direct activation of pertussis toxin-sensitive G proteins by LPA. LPA stimulated a strong increase in the specific phosphorylation of the mitogen-activated protein kinase (immunoblot analysis) that was prevented by the MEK inhibitor PD-98059. Finally, LPA triggered a 2-fold increase in DNA synthesis ([3H]thymidine incorporation) and a 2-fold increase in B lymphoblast number and evoked a 20- to 50-fold increase in immunoglobulin formation. By RT-PCR we detected specific mRNA transcripts for the recently cloned human LPA receptor. Thus our data suggest that LPA behaves as a B cell growth factor.
Antibody Formation↗
Association between a polymorphism in the G protein beta3 subunit gene and lower renin and elevated diastolic blood pressure levels.
Gi proteins mediate the intracellular effects of many vasoactive and proliferative stimuli. Recently such signaling was found to be enhanced in cultured cells of some hypertensive subjects. A polymorphism at position 825 (C-->T) of the G protein beta3 subunit gene (GNB3) was strictly related to this phenotype. The aim of the present investigation was to test the association between this polymorphism and blood pressure and plasma renin levels in humans. A population-based sample (n=608) was analyzed by questionnaire and characterized for blood pressure; plasma renin, prorenin, and aldosterone levels; and Gbeta3 C825T allele status. In individuals without antihypertensive medication (n=474; age range, 52 to 67 years), the polymorphism was mildly associated with diastolic blood pressure (CC: 88.6+/-0.3 mm Hg, n=218; versus CT: 90.1+/-0.7 mm Hg, n=209; versus TT: 91.8+/-1.7 mm Hg, n=47; P=0.02 for trend) but not with systolic blood pressure. Furthermore, the 825T allele was also significantly associated with lower renin and prorenin levels, whereas the aldosterone to renin ratio was elevated in these subjects. Significant associations between the 825T allele and diastolic blood pressure, plasma renin, and prorenin levels (inverse), and the aldosterone to renin ratio persisted after adjustment for age, gender, body mass index, and systolic blood pressure. Finally, when the entire sample was considered and an adjustment was made for covariates, the presence of arterial hypertension and the use of antihypertensive medication were both 1. 8-fold higher in the TT than in the CC genotype group (P<0.05 and P=0.06, respectively). This observation, if replicated in further studies, suggests a molecular mechanism that unifies a higher diastolic blood pressure, a lower renin level, and an elevated aldosterone to renin ratio, ie, a combination of features frequently found in patients with arterial hypertension.
Aged↗
Signalling properties of lysophosphatidic acid in primary human skin fibroblasts: role of pertussis toxin-sensitive GTP-binding proteins.
We have investigated the signalling properties of the naturally occurring intercellular signalling molecule lysophosphatidic acid (LPA) in primary human skin fibroblasts. LPA stimulated phospholipase C activity resulting in the formation of inositol 1,4,5-trisphosphate (IP3) which was accompanied by a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was subject to homologous desensitisation but not to heterologous desensitisation by sphingosine-1-phosphate. The half-maximal effect of LPA on the rise in [Ca2+]i was attained at 7-20 nM. IP3 formation and Ca2+ mobilisation were highly pertussis toxin (PTX)-sensitive (100% and 75%, respectively). LPA also inhibited forskolin-stimulated formation of cAMP, which was partially reversed (51%) when fibroblasts were pretreated with PTX. To directly test the involvement of guanine nucleotide-binding regulatory proteins (G proteins), LPA-induced binding of the stable GTP analogue GTP gamma S was measured. LPA induced an increase in GTP gamma S binding, which was completely inhibited by PTX, implicating the involvement of Gi-type G proteins in LPA signalling. Furthermore, LPA increased DNA synthesis and cell proliferation. Finally, LPA induced the migration of human skin fibroblasts, which in conjunction with the stimulation of cell growth strengthens the presumed involvement of LPA in wound healing and tissue regeneration. Both effects (cell growth and migration) were almost completely PTX-sensitive. Overall, these investigations in primary cultures of human skin fibroblasts confirm and extend our knowledge about LPA signalling, suggesting a pivotal role of receptor coupled activation of Gi-type proteins at least in this cell type.
Calcium↗
Acute saline infusion induces extracellular acidification and activation of the Na+/H+ exchanger in man.
The effect of acute expansion of the extracellular fluid volume (ECV) with isotonic (0.9%) saline on the activity of the lymphocyte Na+/H+ antiport (NHE) was studied in a total of 18 healthy volunteers. Saline was infused at a constant rate so that 4 mmol kg-1 b.w. was administered over 2 h. NHE activity was measured by quantifying cytosolic pH (pHi) recovery following acidification of the cells with propionic acid and by pH clamping at various pHi values between 7.2 and 5.8 using nigericin. Both methods demonstrate NHE activation associated with intravenous saline infusion, the kinetic difference being a marked decrease in the Hill coefficient n from 3.28 +/- 0.21 (SEM) to 2.22 +/- 0.11 in the absence of changes in baseline pHi (7.14 +/- 0.02 vs. 7.08 +/- 0.02; P = 0.15), Vmax (42.8 +/- 2.7 vs. 48.1 +/- 2.8 mmol L-1 min-1; P = 0.08) and pK (6.32 +/- 0.04 vs. 6.35 +/- 0.02). NHE activation was associated with significant decreases in serum chloride (P = 0.016), calcium (P = 0.008), total cholesterol (P = 0.008), low-density lipoproteins (P = 0.016) and high-density lipoproteins (P = 0.008). Moreover, saline infusion induced extracellular acidification with a decrease in pH from 7.39 +/- 0.01 to 7.37 +/- 0.01 (P = 0.016), HCO3- from 23.3 +/- 0.43 mmol L-1 to 21.3 +/- 0.25 mmol L-1 (P = 0.008) and base excess from -1.03 +/- 0.38 mmol L-1 to -3.00 +/- 0.31 mmol L-1 (P = 0.008). Our results show for the first time that acute ECV expansion with isotonic saline is followed by an activation of the lymphocyte NHE. The underlying mechanism(s) remain to be investigated. However, the demonstration in our study of marked changes in acid-base balance induced by acute saline points to a possible inter-relationship of antiporter activation and extracellular acidification.
Acid-Base Equilibrium↗
Prevalence of increased intracellular signal transduction in immortalized lymphoblasts from patients with essential hypertension and normotensive subjects.
OBJECTIVE: To determine the prevalence of enhanced signal transduction in immortalized B lymphoblasts from normotensive subjects and patients with essential hypertension. METHODS: We established Epstein-Barr virus-immortalized lymphoblast cell lines from 26 normotensive and 37 hypertensive subjects. Subsequently, we quantified rises in the cytosolic free Ca2+ concentration, [Ca2+]i, evoked by 0.1 micromol/l platelet-activating factor (PAF) in Fura-2-loaded cells. RESULTS: PAF-induced [Ca2+]i rises were independent of donor age in cells from normotensive and hypertensive subjects. Baseline values of [Ca2+]i were not significantly different in the two groups. Using the mean + 2SD of the PAF-evoked rises in [Ca2+]i above basal (110 nmol/l) as the upper normal value, we estimate that enhanced [Ca2+]i rises are distinctly more prevalent in hypertensive subjects (27%) than they are in normotensive subjects (4%). Similarly, upon definition of normal values by the 99% confidence interval (75 nmol/l), 19% of cells from normotensive versus 43% from hypertensive subjects display enhanced intracellular signaling. CONCLUSION: Enhanced intracellular signal transduction could be the primary defect in approximately one-third of the overall population with essential hypertension.
Adult↗
Sodium-lithium countertransport.
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Animals↗
G proteins, hypertension, and coronary heart disease--novel findings and hypotheses.
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Animals↗
Genetically fixed enhanced G protein activation in essential hypertension.
Recent studies have shown that enhanced Na/H exchanger activity, a frequently observed abnormality in essential hypertension, persists in Epstein-Barr-virus-immortalised lymphoblasts from afflicted patients. This phenomenon is associated with an enhanced proliferation of these cell lines. Studies have ruled out structural changes in the Na/H exchanger protein, but demonstrated a distinct enhancement of intracellular signal transduction in these cell lines. The ultimate cause can be assigned to an obviously genetically fixed increased reactivity of pertussis-toxin-sensitive G proteins, which can easily explain the increased formation of intracellular- second messengers, enhanced Ca2+ signals, and enhanced proliferation of these cell lines. Thus, hypertension in the subset of patients with enhanced Na/H exchanger activity could be caused by increased G protein reactivity.
Animals↗
Selectively enhanced cellular signaling by Gi proteins in essential hypertension. G alpha i2, G alpha i3, G beta 1, and G beta 2 are not mutated.
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 Gs with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of G alpha i2, G alpha i3, G beta 2, and G beta 4. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits G alpha i2, G alpha i3, G beta 1, and G beta 2 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.
Base Sequence↗
Potential effect of metabolic acidosis on beta 2-microglobulin generation: in vivo and in vitro studies.
Beta 2-microglobulin (beta 2M) is responsible for dialysis-associated amyloidosis. Level of beta 2M in plasma increase during chronic renal failure; however, retention does not appear to be the sole mechanism responsible. The effect of metabolic acidosis on beta 2M production was examined. Thirty-six patients with stable chronic renal insufficiency, 12 uremic patients before their first dialysis, 8 hemodialysis patients who were assigned to acetate or bicarbonate dialysate and then crossed over to the alternative regimen, and 6 normal subjects given NH4Cl to initiate metabolic acidosis were studied. In vitro studies in the human myeloid cell line U 937 were also performed. beta 2M protein was measured with ELISA, beta 2M mRNA was measured with reverse transcription polymerase chain reaction, and the U 937 cells were studied at two pH levels with FACScan flow cytometry. The cells were exposed in vitro up to 60 min in a buffered incubation medium to either pH 5.10 or pH 7.34. An inverse correlation was found between beta 2M and bicarbonate concentrations in plasma in the stable chronic renal failure patients (r = -0.54; P < 0.05) and in the uremic patients before their first dialysis (r = -0.72; P < 0.05). In hemodialysis patients, blood pH and plasma bicarbonate values were lower (P < 0.05) and beta 2M concentrations in plasma were higher (P < 0.05) with acetate than with bicarbonate dialysate. In normal men, NH4Cl resulted in an increase (P < 0.05) in beta 2M mRNA expression in lymphocytes by an average factor of 1.5 (range, 1.1 to 1.8). In U 937 cells, the cell surface expression of beta 2M and HLA Class I heavy chain assembled with beta 2M decreased at low pH compared with normal pH. Concomitantly, an increase in beta 2M release into the supernatant was observed, possibly as the result of beta 2M dissociation from cell surface HLA Class I complex. The results suggest that metabolic acidosis may enhance cellular beta 2M generation and release.
Acidosis↗