Dieter Rosskopf

Membrane sodium-proton exchange and primary hypertension.

Recent studies have revealed that an enhancement of sodium-proton exchange is a frequently observed ion transport abnormality in essential hypertension. An altered antiport activity not only is measurable in blood cells of hypertensive subjects ex vivo but also is detectable in skeletal muscle in vivo. Several lines of argument suggest that the altered antiport activity is not an epiphenomenon of hypertension: 1) the increased activity is found only in a subgroup of patients with high blood pressure, 2) it is not tightly correlated to the severity or duration of hypertension, and 3) high sodium-proton exchange activity persists over time and is not affected by antihypertensive treatment. Available evidence suggests that enhanced sodium-proton exchange is associated with or a cause for the structural alterations found in resistance vessels of hypertensive individuals (media hypertrophy) and left ventricular hypertrophy. This review summarizes some of the physiological properties and roles of the sodium-proton exchanger and discusses its kinetic properties in essential hypertension. Furthermore, the reasons for the enhanced antiport activity and its potential implications regarding the pathogenesis of hypertension are discussed.

Selectively Enhanced Cellular Signaling by Gi Proteins in Essential Hypertension

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.

Enhanced Immunoglobulin Formation of Immortalized B Cells From Hypertensive Patients

Increased immunoglobulin levels and leukocyte counts have frequently been reported in essential hypertension. The underlying mechanisms, however, have remained obscure. Enhanced Na(+)-H+ exchanger activity is another frequently observed abnormality in essential hypertension that persists in immortalized B lymphoblasts and coincides with enhanced proliferation. We investigated the capacity of B lymphoblasts from essential hypertensive patients to synthesize and secrete immunoglobulins. Six B cell lines from essential hypertensive patients with enhanced Na(+)-H+ exchanger phenotype and six cell lines from normotensive subjects were studied. Lymphocyte markers were visualized by immunostaining. Immunoglobulin secretion was analyzed by enzyme-linked immunosorbent assay. These cell lines did not differ with respect to B cell markers. In response to 100 nmol/L platelet-activating factor, cells from hypertensive patients proliferated distinctly more quickly and their cell number increased by 3.9 +/- 0.4-fold (mean +/- SD) within 4 days, whereas the number of cells from normotensive subjects increased by only 2.6 +/- 0.1-fold. Furthermore, platelet-activating factor induced average increases in IgM and IgG formation of 13.3- and 5.4-fold, respectively, in lymphoblasts from hypertensive patients, which was significantly higher than increases in cells from normotensive subjects (1.4- and 1.2-fold, respectively). Thus, lymphoblasts from hypertensive patients proliferate more quickly and secrete more immunoglobulins in response to a physiological stimulus in vitro. This may contribute to the raised immunoglobulin levels and leukocyte counts reported in vivo.

Detection of Na+/H+ exchanger mRNA in human neutrophils and lymphocytes using the polymerase chain reaction

Using the reverse polymerase chain reaction (RT‐PCR), we have examined the expression of Na+/H+ exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection or low abundance Na+/H+ antiporter specific transcripts by RT‐PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na+/H+ exchanger mRNA expression in human cells and tissue samples.

Expression, functional characterization and tissue distribution of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE).

Abstract 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 µmol/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.

Expression and Activity of the Na+/H+ Exchanger NHE-1 in Various Tissues of Spontaneously Hypertensive Rats and Normotensive Wistar-Kyoto Rats

Vascular smooth muscle cells (VSMC) from the spontaneously hypertensive rat (SHR) exhibit an enhanced Na+/H+ exchange activity which coincides with increased proliferation. The aim of the present work was to investigate Na+/H+ exchange activity in VSMC and thymocytes from SHR and Wistar-Kyoto (WKY) rats and to examine the steady state levels of specific Na+/H+ exchanger (isoform 1; NHE-1) mRNA transcripts in VSMC, thymocytes, heart, and kidney.VSMC from SHR proliferated significantly faster than those from WKY rats. NaVH+ exchange activity (Vmax) was significantly (p + × liter–1 × min–1; mean ± SE). In contrast, Na+/H+ exchange activity was higher in thymocytes from WKY rats. By Northern blot analysis, we observed similar steady state mRNA levels for NHE-1 in tissues from SHR and WKY, respectively.We conclude that the enhanced Na+/H+ exchange activity found in VSMC from SHR is not expressed in all tissues of SHR. Furthermore, our data suggest that over-expression of NHE-1 is not responsible for the enhanced Na+/ H+ exchange activity in VSMC from SHR. Thus, alterations in cell signalling located upstream of the Na+/H+ exchanger may induce both an increased proliferation and an altered post-translational regulation of Na+/H+ exchange activity in VSMC from SHR.

Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)amiloride

We used the radiolabelled inhibitor of Na+/H+ exchange 5-(N-methyl-N-[3H]isobutyl)amiloride ([3H]-MIA) for assessment of the amount of Na+/H+ exchanger in intact human blood platelets. The inhibition constant, KI, of unlabelled MIA toward the antiport was determined at 100 nM. Washed platelets were incubated for 5 s with different concentrations of [3H]-MIA in the presence or absence of an excess concentration of unlabelled amiloride (400 microM). The platelets were rapidly centrifuged and the radioactivity in the pellet was determined. Scatchard analysis revealed one single class of specific binding sites (KD = 63 nM) and a maximum binding capacity of 500 sites/cell. The turnover rate of the Na+/H(+)-exchanger in unstimulated platelets was estimated at 800/s at 25 degrees C.

HOE 694 Blocks Na+/H+ Exchange in Human B Lymphoblasts without Influencing Proliferation

The effect of HOE 694 (3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride), a novel inhibitor of Na+/H+ exchange, on pHi recovery from acidification and on proliferation of human B lymphoblasts was investigated. HOE 694 inhibited the Na+/H+ exchanger in a concentration-dependent manner. Inhibition constants (Ki) were calculated at 89 ± 5 and 54 ± 7 nmol/l (means ± SD; n = 5) at 135 and 79 mmol/l external Na+, respectively. This indicates a competition of HOE 694 with external Na+ ions at a common binding site of the Na+/H+ exchanger. Subsequently, the effects of HOE 694 upon DNA synthesis and proliferation of lymphoblasts were examined. HOE 694 inhibited cell proliferation and DNA synthesis only at concentrations of > 10 µmol/l. This suggests that in the presence of bicarbonate a functionally active Na+/ H+ exchanger is not required for the proliferation of B lymphoblasts.