Molecular basis of human salt sensitivity: the role of the 11beta-hydroxysteroid dehydrogenase type 2.

Salt-sensitive subjects (SS) increase their blood pressure with increasing salt intake. Because steroid hormones modulate renal sodium retention, we hypothesize that the activity of the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme is impaired in SS subjects as compared with salt-resistant (SR) subjects. The 11betaHSD2 enzyme inactivates 11-hydroxy steroids in the kidney, thus protecting the nonselective mineralocorticoid receptor from occupation by glucocorticoids. We performed an association study using a recently identified single AluI polymorphism in exon 3 and a polymorphic microsatellite marker of the HSD11B2 gene in 149 normotensive white males (37 SS and 112 SR). The activity of the enzyme 11betaHSD2 was assessed by determining the urinary ratio of cortisol (THF+5alphaTHF) to cortisone (THE) metabolites by gas chromatography in all the 37 SS subjects and in 37 age- and body habitus-matched SR volunteers. Mean (THF+5alphaTHF)/THE ratio was markedly elevated in SS subjects compared with SR subjects (1.51 +/- 0.34 vs. 1.08 +/- 0.26, P < 0.00001), indicating enhanced access of glucocorticoids to the mineralocorticoid receptor in SS subjects. In 58% of SS subjects this ratio was higher than the maximum levels in SR subjects. The salt-induced elevation in arterial pressure increased with increasing (THF+5alphaTHF)/THE ratio (r2 = 0.51, P < 0.0001). A total of 12 alleles of the polymorphic microsatellite marker were detected. Homozygosity for the allele A7 was higher in SS subjects than in SR subjects (41 vs. 28%, P < 0.005), whereas the occurrence of the allele A7 with allele A8 was lower in SS subjects than in SR subjects (8 vs. 15%, P < 0.03). The prevalence of salt sensitivity was 35% in subjects with allele A7/A7, whereas salt sensitivity was present in only 9% of the subjects with allele A7/A8. The (THF+5alphaTHF)/THE ratio was higher in subjects homozygous for the A7 microsatellite allele as compared with the corresponding control subjects. The prevalence of the AluI allele was 8.0% in SR subjects and 5.4% in SS subjects and did not correlate with blood pressure. The decreased activity of the 11betaHSD2 in SS subjects indicates that this enzyme is involved in salt-sensitive blood pressure response in humans. The association of a polymorphic microsatellite marker of the gene with a reduced 11betaHSD2 activity suggests that variants of the HSD 11B2 gene contribute to enhanced blood pressure response to salt in humans.

Fiber-type-specific expression of essential (alkali) myosin light chains in human skeletal muscles.

We studied the expression patterns of the essential (alkali) myosin light-chain isoforms in adult human skeletal muscles, using in situ hybridization and single-fiber protein analysis. In analogy to other species, we found that the fiber type-specific expression of essential myosin light chains is regulated via the availability of the respective mRNAs in a given fiber. In contrast to other species, the slow isoform 1sa was only expressed in the most oxidative Type I fibers (Subtype IA) in addition to 1sb. These fibers also contained high levels of carbonic anhydrase III. Within the fibers, the essential myosin light-chain mRNAs were located preferentially in the perinuclear regions and to a lesser extent in the intermyofibrillar spaces, a distribution that excludes cotranslational assembly of these light chains into the myofibrils as the main mechanism. In comparing leg and shoulder muscles, we found less distinct fiber typing in the expression patterns of the essential myosin light chains in the leg muscles than in muscles from the shoulder region.

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Multiple endocrine neoplasia type 1 (MEN1) gene mutations in a subset of patients with sporadic and familial primary hyperparathyroidism target the coding sequence but spare the promoter region.

Germ line mutations of the multiple endocrine neoplasia type 1 (MEN1) tumour suppressor gene cause MEN1, a rare familial tumour syndrome associated with parathyroid hyperplasia, adenoma and hyperparathyroidism (HP). Here we investigated the role of the MEN1 gene in isolated sporadic and familial HP. Using RT-PCR single-strand conformational polymorphism screening, somatic (but not germ line) mutations of the MEN1 coding sequence were identified in 6 of 31 (19.3%) adenomas from patients with sporadic primary HP, but none in patients (n=16) with secondary HP due to chronic renal failure. MEN1 mutations were accompanied by a loss of heterozygosity (LOH) for the MEN1 locus on chromosome 11q13 in the adenomas as detected by microsatellite analysis. No DNA sequence divergence within the 5' region of the MEN1 gene, containing the putative MEN1 promoter, was detectable in HP adenomas. Clinical characteristics were not different in HP patients with or without MEN1 mutation. Heterozygous MEN1 gene polymorphisms were identified in 9.6% and 25% of patients with primary and secondary HP respectively. In a large kindred with familial isolated familial HP, MEN1 germ line mutation 249 del4 and LOH was associated with the HP phenotype and a predisposition to non-endocrine malignancies. We suggest that the bi-allelic somatic loss of MEN1 wild-type gene expression is involved in the pathogenesis of a clinically yet undefined subset of sporadic primary HP adenomas. MEN1 genotyping may further help define the familial hyperparathyroidism-MEN1 disease complex, but it seems dispensable in sporadic primary HP.

Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-gamma-dependent way.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARgamma ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARgamma activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARgamma-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARgamma in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARgamma activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.

The use of 33P-labelled riboprobes for in situ hybridizations: localization of myosin alkali light-chain mRNAs in adult human skeletal muscle.

33P-labelled probes were used to localize the mRNAs coding for the myosin alkali light-chain isoforms MLC 1f/3f and MLC 1sb in adult human muscles, which are distributed in characteristic fibre type specific patterns. In situ hybridizations of 33P-labelled probes were compared with probes carrying 35S or digoxigenin labels. Signals of equal strength were obtained with each of the three labels. The preferentially peripheral localization of these mRNAs in the muscle fibres could be clearly seen with all three probes, with digoxigenin probes providing the best resolution. 33P can serve as a viable alternative in this type of experiment. These experiments with adult human muscles also showed that the post mortem stability of RNA in human muscle is better than generally assumed. We could detect no signs of degradation in RNA prepared from heart ventricle as well as skeletal muscle up to 24 hours post mortem. In situ hybridizations worked equally well in biopsy material as in post mortem samples.

An efficient polymerase chain reaction approach for the quantitation of multiple RNAs in human tissue samples.

We describe a PCR quantitation approach that we have set up to study gene expression in human skeletal muscle biopsies. It is characterized by the independent standardization of the individual steps and ignores attempts to control for the efficiency of the PCR. Different RNA extraction/reverse transcription efficiencies are normalized by the addition of an unrelated cRNA. Quantitation is achieved by parallel amplifications of reference samples containing known amounts of PCR products. Precision was achieved by multiple measurements of several samples. Our approach allows for the detection of less than twofold differences (27-88%, depending on the RNA species studied) among samples. A comparison of biopsies from highly trained endurance runners with biopsies from untrained subjects showed that the increased mitochondrial density in the runners' samples is accompanied by a proportional increase in the concentration of the mRNA of cytochrome c oxidase subunit IV.

mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes.

Improvements in endurance capacity by training are associated with structural and biochemical adaptations of working muscles that affect the mitochondrial compartment. We investigated whether the 1.8-fold higher mitochondrial volume density in a group of endurance-trained athletes compared with untrained subjects was reflected by higher steady-state levels of mRNAs coding for components of the oxidative phosphorylation pathway using a quantitative polymerase chain reaction approach. We found that mitochondrially encoded RNAs (cytochrome-c oxidase subunit I, NADH reductase subunit 6, 16S rRNA), as well as nuclear-encoded RNAs (cytochrome-c oxidase subunit IV, succinate dehydrogenase, fumarase) are all increased coordinately in the athletes (1.54- to 1.94-fold). In addition, mitochondrial (mt) DNA concentration was also 1.55-fold higher in the trained athletes, whereas genomic DNA was not changed. Our findings thus show similar RNA expression of mitochondrially encoded genes in sedentary and endurance-trained subjects, whereas pretranslational control mechanisms account for higher levels of nuclear-encoded RNAs in the athletes.

Expression of fos and jun genes in human skeletal muscle after exercise.

It is believed that the induction of the fos and jun gene family of transcription factors might be at the origin of genetic events leading to the differential regulation of muscle-specific genes. We have investigated the effect of a 30-min running bout in untrained subjects on the expression of the mRNAs of all members of the fos and jun gene families, including c-fos, fosB, fosBdel, fra-1, and fra-2 as well as c-jun, junB, and junD. While the fos family members were transiently upregulated 10- to 20-fold (an exception being fra-2) the induction of the jun family members was up to 3-fold only. The induction of c-fos could also be demonstrated at the protein level. Both c-fos and c-jun mRNAs were coinduced in muscle fiber nuclei. The induction was not restricted to a particular fiber type, as expected from established muscle fiber recruitment schemes, but followed a "patchy" pattern confined to certain regions of the muscle. The signals leading to the expression of these immediate early genes are therefore unclear.