Liver mitochondrial DNA damage and genetic variability of Cytochrome b - a key component of the respirasome - drive the severity of fatty liver disease.

BACKGROUND AND AIMS: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis - NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). METHODS: We included 252 liver specimens of NAFLD patients - in whom histological disease ranged from mild to severe - which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal - a product of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage - were measured. RESULTS: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103  bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. CONCLUSION: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects.

Systematic review with meta-analysis: the significance of histological disease severity in lean patients with nonalcoholic fatty liver disease.

BACKGROUND: Current evidence suggests that lean and obese patients with nonalcoholic fatty liver disease (NAFLD) share an altered metabolic and cardiovascular profile. However, there is an incomplete understanding of the natural history of "lean-NAFLD." Indeed, an unanswered question is whether lean (BMI ≤ 25 Kg/m2 ) NAFLD-patients are protected from severe histological outcomes. AIM: To perform a meta-analysis with the goal of providing a quantitative estimation of the magnitude of fibrosis, as well as histological features associated with the disease severity, in lean versus overweight/obese-NAFLD patients. METHODS: Through a systematic search up to July 2017, we identified eight studies that compared histological outcomes in lean (n = 493) versus overweight/obese (n = 2209) patients. RESULTS: Relative to lean-NAFLD, overweight/obese-NAFLD patients showed significantly (P = .032) higher fibrosis scores; the observed difference in means between the two groups, which is the absolute difference between the mean value of fibrosis score [0-4] ± standard error, was 0.28 ± 0.13. The risk of having nonalcoholic steatohepatitis-NASH (OR 0.58 95% CI 0.34-0.97) was significantly lower in lean-NAFLD (n = 322) than in overweight/obese-NAFLD (n = 1357), P = .04. Relative to lean-NAFLD, overweight/obese-NAFLD patients also have significantly greater NAFLD activity (difference in means ± SE: 0.58 ± 0.16, P = .0004) and steatosis (difference in means ± SE: 0.23 ± 0.07, P = .002) scores. CONCLUSIONS: Lean-NAFLD patients tend to show less severe histological features as compared to overweight/obese-NAFLD patients. Subsequent longitudinal assessment is needed to understand the clinical impact of these findings; however, the significant ~ 25% increment of mean fibrosis score in overweight/obese patients suggests that obesity could predict a worse long-term prognosis.

Systematic review with meta-analysis: risk factors for non-alcoholic fatty liver disease suggest a shared altered metabolic and cardiovascular profile between lean and obese patients.

BACKGROUND: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is closely associated with the co-occurrence of multiple pathological conditions characterising the metabolic syndrome (MetS), obesity in particular. However, NAFLD also develops in lean subjects, whose risk factors remain poorly defined. METHODS: We performed a meta-analysis of 15 studies, along with the data pertaining to our own population (n=336 patients). Data from lean (n=1966) and obese (n=5938) patients with NAFLD were analysed; lean (n=9946) and obese (n=6027) subjects without NAFLD served as controls. RESULTS: Relative to the lean non-NAFLD controls, lean patients with NAFLD were older (3.79±0.72 years, P=1.36×10-6 ) and exhibited the entire spectrum of the MetS risk factors. Specifically, they had a significant (P=10-10 ) increase in plasma glucose levels (6.44±1.12 mg/dL) and HOMA-IR (0.52±0.094-unit increment), blood lipids (triglycerides: 48.37±3.6, P=10-10 and total cholesterol: 7.04±3.8, mg/dL, P=4.2×10-7 ), systolic (5.64±0.7) and diastolic (3.37±0.9) blood pressure (mm Hg), P=10-10 , and waist circumference (5.88±0.4 cm, P=10-10 ); values denote difference in means±SE. Nevertheless, the overall alterations in the obese group were much more severe when compared to lean subjects, regardless of the presence of NAFLD. Meta-regression suggested that NAFLD is a modifier of the level of blood lipids. CONCLUSION: Lean and obese patients with NAFLD share a common altered metabolic and cardiovascular profile. The former, while having normal body weight, showed excess of abdominal adipose tissue as well as other MetS features.

The impact of hypertension on leukocyte telomere length: a systematic review and meta-analysis of human studies.

Shortened leukocyte telomere length (LTL) is a novel biomarker for age and age-related diseases. Several epidemiological studies have examined the association between telomere length in surrogate tissues (for example, blood cells) and hypertension, and meanwhile the majority of studies reported an association some individual studies do not. We carried out a systematic review and meta-analysis to address the hypothesis that, in humans, telomere length is related with hypertension. Searches were conducted in Pubmed by September 2015 and reference lists of retrieved citations were hand searched. Eligible studies measured telomeres for both hypertensive and normotensive subjects. No restrictions were placed on sample size, publication type, age or gender. We calculated summary estimates using fixed and random effects meta-analysis. Publication bias and heterogeneity among studies were further tested. Meta-analyses from 3097 participants (1415 patients with hypertension and 1682 control subjects) showed a significant standardized mean difference between LTL in hypertensive patients and controls, either in the fixed (P<5 × 10-6) or the random model (P<0.005). Heterogeneity among studies was substantial (Q-statistic P-value <0.001, I2 97.73%). Sensitivity analysis indicated that no single study changed the standardized mean difference qualitatively (0.022> random model P-value >0.002). Egger's test for asymmetry of effect sizes (intercept±s.e.=-7.278±3.574; P=0.072) did not show evidence for strong study publication bias. Leukocyte telomeres may be shorter in hypertensive than in normotensive individuals. Larger studies controlling for confounder effects are needed to confirm these findings and further explore sources of heterogeneity.

Mendelian randomisation suggests no beneficial effect of moderate alcohol consumption on the severity of nonalcoholic fatty liver disease.

BACKGROUND: Previous epidemiological studies suggest that patients diagnosed with nonalcoholic fatty liver disease (NAFLD) who drink light to moderate amounts of alcohol (up to ~30 g per day) have less severe histological lesions compared with nondrinkers. However, while the cross-sectional nature of current evidence precludes assessment of causality, cumulative lifetime-exposure of moderate alcohol consumption on histological outcomes has never been evaluated. AIM: To overcome these limitations, a Mendelian randomisation study was performed using a validated genetic variant (rs1229984 A;G) in the alcohol dehydrogenase (ADH1B) gene as a proxy of long-term alcohol exposure. METHODS: We first assessed whether the instrumental variant (rs1229984) was associated with the amount of alcohol consumption in our cohort. We further explored the association between the variant and histological outcomes; a sample of 466 individuals, including 266 patients with NAFLD confirmed by liver biopsy, was studied. RESULTS: We found that carriers of the A-allele consumed significantly lower amounts of alcohol compared with noncarriers (2.3 ± 5.3 vs. 8.18 ± 21 g per day, mean ± s.d., P = 0.03). The analysis of association with the disease severity showed that carriers of the A-allele had lower degree of histological steatosis (1.76 ± 0.83 vs. 2.19 ± 0.78, P = 0.03) and lower scores of lobular inflammation (0.54 ± 0.65 vs. 0.95 ± 0.92, P = 0.02) and NAFLD-Activity Score (2.9 ± 1.4 vs. 3.7 ± 1.4, P = 0.015) compared with noncarriers. CONCLUSION: Mendelian randomisation analysis suggests no beneficial effect of moderate alcohol consumption on NAFLD disease severity.

The influence of common gene variants of the xenobiotic receptor (PXR) in genetic susceptibility to intrahepatic cholestasis of pregnancy.

BACKGROUND: The xenobiotic nuclear pregnane X receptor is implicated in many physiological pathways and diseases, including bile acid detoxification and cholestasis. Aim To estimate the contribution of common gene variants of the xenobiotic receptor (pregnane X receptor, PXR) to genetic susceptibility to intrahepatic cholestasis of pregnancy. METHODS: A total of 101 intrahepatic cholestasis of pregnancy patients and 171 healthy pregnant women in the third trimester of their pregnancies were included. Four tag single nucleotide polymorphisms (SNPs) (rs12488820 C/T, rs2472671 C/T, rs2461823 A/G, and rs1054191 A/G) encompassing 36 kb in chromosome 3, with a minor allele frequency > or =0.10 and representing 33 polymorphic sites were genotyped. Besides these, three additional SNPs (rs3814057, rs6785049, and rs7643645) were included because they showed previous evidence of functionality. RESULTS: Genotypic test for single SNPs showed that rs2461823 genotypes were significantly associated with intrahepatic cholestasis of pregnancy (P < 0.0069), OR per G allele: 1.44, 95% CI: 1.01-2.05, P < 0.042. The Cochran-Armitage test for trend and the allelic test showed a significant association with disease status (P < 0.04 and 0.03 respectively), G being the risk allele. A positive association between rs2461823 and ALT, AST, and bilirubin concentrations was observed. Neonate birth weight adjusted by the Capurro index was significantly associated with rs2461823 (P < 0.05); the proportion of the total variation attributed to rs2461823 genotypes was 7.8%. CONCLUSION: Common PXR polymorphisms may contribute to the genetic susceptibility to intrahepatic cholestasis of pregnancy.

Role of the C-344T aldosterone synthase gene variant in left ventricular mass and left ventricular structure-related phenotypes.

AIM: We performed a systematic review of the literature by means of a meta-analysis to evaluate the influence of the aldosterone synthase gene (CYP11B2) C-344T polymorphism on left ventricular mass (LVM) and related phenotypes. DESIGN: From 485 reports, we included 14 studies about the association between the C-344T variant and left ventricular mass and left ventricular structure-related phenotypes, from which information about number of subjects in each category, outcomes data and genotyping performed with a validated molecular method could be extracted. Fixed and random effect models were used to pool data from individual studies, and the results in the abstract show the extreme genotype comparison, homozygous TT vs homozygous CC. RESULTS: From a total of 2157 subjects, we found no significant association between LVM and the C-344T variant (D: 0.049, 95% CI: 0.091 to 0.179, p = 0.462). Similarly, no significant association was found for interventricular septal-wall thickness (D: 0.027, 95% CI: -0.090 to 0.143, p = 0.654, n: 2105). However, homozygous TT hypertensive subjects had increased LVM (D: 0.251, 95% CI: 0.020 to 0.481, p = 0.04, n: 332). Lastly, in 10 homogeneous studies posterior wall thickness (PWT) was lower in homozygous CC individuals (D: 0.142, 95% CI: 0.016 to 0.268, p = 0.028, n = 1994). CONCLUSION: Independently of hypertension, homozygous individuals for the -344T allele may have 2.4% higher PWT compared to homozygous subjects for the C-344 allele. Besides, homozygous hypertensive TT subjects show a 6.9% increase in LVM compared to CC homozygous subjects.

Effects of rotating shift work on biomarkers of metabolic syndrome and inflammation.

OBJECTIVE: The major function of the circadian system is the internal cycling of physiological and metabolic events. The present study sought to explore the effect of rotating shift work schedule on leucocyte count and its relationship with risk factors of metabolic syndrome (MS). DESIGN AND PARTICIPANTS: From a population-based design, 1351 men of self-reported European ancestry were included in a cross-sectional study: 877 day workers were compared with 474 rotating shift workers. Medical history, health examination including anthropometric and arterial blood pressure measurements, a questionnaire on health-related behaviours and biochemical determinations was given to all participants. RESULTS: In comparison with day workers, rotating shift workers had elevated (mean +/- SE) body mass index (27.1 +/- 0.3 vs. 26.3 +/- 0.2, P < 0.0154), waist-hip ratio (0.95 +/- 0.01 vs. 0.93 +/- 0.01, P < 0.00024), diastolic arterial blood pressure (78 +/- 1 vs. 76 +/- 1, P < 0.033), fasting insulin (65.5 +/- 2.9 vs. 55.9 +/- 1.9 pmol L(-1), P < 0.017), Homeostasis Model Assessment index (2.12 +/- 0.11 vs. 1.77 +/- 0.07, P < 0.0027), triglycerides (1.71 +/- 0.1 vs. 1.5 +/- 0.1 mmol L(-1), P < 0.002), uric acid (292.7 +/- 2.8 vs. 282 +/- 3.4 micromol L(-1), P < 0.01) and leucocyte count (7030 +/- 84 vs. 6730 +/- 58, P < 0.0094). In multiple regression analysis, leucocyte count was correlated with rotating shift work independently of age, smoking, education and components of MS. CONCLUSION: The odds ratio for MS in rotating shift workers compared with day workers was 1.51 (95% CI 1.01-2.25), independently of age and physical activity. Increased leucocyte count, a biological marker of systemic inflammation, was associated with rotating shift work.

Peroxisome proliferator-activated receptor gamma and its coactivator-1 alpha may be associated with features of the metabolic syndrome in adolescents.

Our objective was to search for differences in genotypes of peroxisome proliferator-activated receptor gamma (PPARgamma) (Pro12 Ala) and its coactivator PGC-1alpha (Gly482 Ser) in adolescents harboring features of metabolic syndrome. In a population-based study, we determined medical history, anthropometric variables, biochemical measurements and arterial blood pressures of 934 high-school students of Caucasian origin. We selected 220 adolescents who had systolic or diastolic blood pressures more than the 80th or less than the 20th percentiles based on the previous single set of measurements. One hundred and seventy-five adolescents completed the study and underwent two additional blood pressure measurements on different days, as well as biochemical analysis and genotyping. We found no association between insulin resistance, body mass index (BMI) and leptin levels and PPARgamma and PGC-1alpha genotypes. The 12 Ala PPARgamma allele was associated with increased waist-to-hip ratio (WHR) and carriers seemed to have higher diastolic blood pressure and lower pulse pressure than non-carriers, particularly in the hypertensive and overweight group. Although Ser482 Ser PGC-1alpha homozygotes had lower WHRs than other PGC-1alpha genotypes, they were more frequent in the hypertensive group than in the normotensive (44.4 vs 24.5%, P<0.03), so the 482 Ser PGC-1 allele was in our population a risk factor for hypertension independently of WHR, homeostasis model assessment of insulin resistance, BMI and Pro12 Ala PPARgamma variant (odds ratio=4.0, 95% confidence interval 1.5-10.6, P<0.01). Multiple regression analysis showed that age- and sex-adjusted systolic blood pressure correlated with the 482 Ser PGC-1 allele regardless of those covariates. In conclusion, the Gly482 Ser variant of the PGC-1alpha gene may be an independent genetic risk factor for young-onset hypertension.

Thyrotropin-releasing hormone receptor (TRHR) gene is associated with essential hypertension.

In essential hypertension, a polygenic and multifactorial syndrome, several genes interact with the environment to produce high blood pressure. Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. We have described that TRH overexpression induces hypertension in a normal rat, which was reversed by TRH antisense treatment. This treatment also reduces the central TRH hyperactivity in spontaneously hypertensive rats and normalizes blood pressure. Human TRH receptor (TRHR) belongs to the G protein-coupled seven-transmembrane domain receptor superfamily. Mutations of these receptors may result in constitutive activation. As it has been demonstrated that hypertensive patients have a blunted TSH response to TRH injection, suggesting a defect in the TRHR, we postulate that the TRHR gene is involved in human hypertension. We studied 2 independent populations from different geographic regions of our country: a sample of adult subjects from a referral clinic and a population-based sample of high school students. In search of molecular variants of TRHR, we disclosed that a polymorphic TG dinucleotide repeat (STR) at -68 bp and a novel single nucleotide polymorphism, a G-->C conversion at -221 located in the promoter of the TRHR are associated with essential hypertension. As STRs detected in gene promoters are potential Z-DNA-forming sequences and seem to affect gene expression, we studied the potentially different transcriptional activity of these TRHR promoter variants and found that the S/-221C allele has a higher affinity than does the L/G-221 allele to nuclear protein factor(s). Our findings support the hypothesis that the TRHR gene participates in the etiopathogenesis of essential hypertension.

Antisense inhibition of thyrotropin-releasing hormone reduces arterial blood pressure in spontaneously hypertensive rats.

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.

Expression of TRH and TRH-like peptides in a human glioblastoma-astrocytoma cell line (U-373-MG).

The human glioblastoma-astrocytoma cell line U-373-MG shows morphological features typical of its neuroectodermal origin. Cells showed positive immunostaining for the glial fibrillary acidic protein. We used this cell culture for studying the putative production of TRH and TRH-related peptides. In a cell extract and conditioned medium, cation and anion exchange chromatography and HPLC revealed the presence of TRH and acidic TRH-like peptides which were identified, at least in part, as pGlu-Glu-ProNH(2). These findings demonstrated that U-373-MG cells are able to produce and release these peptides. Further evidence of TRH synthesis was obtained by amplification using RT-PCR of a 396 bp fragment that corresponds to the TRH precursor mRNA. Our results therefore suggest that the U-373-MG cell line may be a useful model for studying the regulation of TRH and TRH-related peptide production and the interaction of these peptides with other classical neurotransmitter systems. In fact, pilocarpine (a muscarinic cholinergic agonist) enhanced and nicotine (a nicotinic cholinergic agonist) decreased TRH and TRH-related compound production by this cell line. These data also point out that glia may produce substances with neuromodulatory action.

[Molecular genetics of essential hypertension. Susceptibility and resistance genes]. / Genética molecular de la hipertensión arterial esencial. Genes de susceptibilidad y resistencia.

Essential Hypertension (EH) is a multifactorial and polygenic syndrome with a high impact in public health. Recently, rare mendelian forms of hypertension such as glucocorticoid-remediable aldosteronism (GRA), apparent mineralocorticoid excess (AME) and Liddle Syndrome caused by single gene mutations have been identified in which the mechanism is an increased sodium retention. Therefore, it is tempting to speculate that the most common forms of EH may be due to diverse highly prevalent molecular variants of susceptibility genes with low penetrance that are involved in arterial blood pressure (ABP) and electrolytic balance. Although a number of candidate genes such as NO synthases, ANP, ion transporters, adducins, LDL receptor, etc. can participate, renin-angiotensin system components are the most extensively studied. Although not associated with EH, the ACE D allele seems to confer a high risk of CHD or LVH. Angiotensinogen 235T and 174M variants are more likely associated with EH and positively correlate with clinical or ambulatory ABP in adolescents or adults. Individuals who carry these angiotensinogen alleles would be at 1.4 higher risk of suffering EH than homozygotes for M235 or T174 alleles. Associations of AT1 receptor variants with EH remain to be definitively defined. In conclusion, the characterization of the genetic background, although difficult at the present time, may have clear benefits in terms of defining a more rational therapy and prevention in individuals at risk. Even though this aim seems difficult to achieve since more than 150 candidate genes have been postulated as the cause of EH, with 6 to 10 SNPs in each of them, new technologies such as DNA micro-arrays will provide us with the opportunity to analyse the total genetic risk in each subject.

Genética molecular de la hipertensión arterial esencial. Genes de susceptibilidad y resistencia. / [Molecular genetics of essential hypertension. Susceptibility and resistance genes]

Essential Hypertension (EH) is a multifactorial and polygenic syndrome with a high impact in public health. Recently, rare mendelian forms of hypertension such as glucocorticoid-remediable aldosteronism (GRA), apparent mineralocorticoid excess (AME) and Liddle Syndrome caused by single gene mutations have been identified in which the mechanism is an increased sodium retention. Therefore, it is tempting to speculate that the most common forms of EH may be due to diverse highly prevalent molecular variants of susceptibility genes with low penetrance that are involved in arterial blood pressure (ABP) and electrolytic balance. Although a number of candidate genes such as NO synthases, ANP, ion transporters, adducins, LDL receptor, etc. can participate, renin-angiotensin system components are the most extensively studied. Although not associated with EH, the ACE D allele seems to confer a high risk of CHD or LVH. Angiotensinogen 235T and 174M variants are more likely associated with EH and positively correlate with clinical or ambulatory ABP in adolescents or adults. Individuals who carry these angiotensinogen alleles would be at 1.4 higher risk of suffering EH than homozygotes for M235 or T174 alleles. Associations of AT1 receptor variants with EH remain to be definitively defined. In conclusion, the characterization of the genetic background, although difficult at the present time, may have clear benefits in terms of defining a more rational therapy and prevention in individuals at risk. Even though this aim seems difficult to achieve since more than 150 candidate genes have been postulated as the cause of EH, with 6 to 10 SNPs in each of them, new technologies such as DNA micro-arrays will provide us with the opportunity to analyse the total genetic risk in each subject.

Parathyroid hormone-related protein expression in vascular smooth muscle of spontaneously hypertensive rats: evidence for lack of response to angiotensin II.

OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.

Central overexpression of the TRH precursor gene induces hypertension in rats: antisense reversal.

Extrahypothalamic TRH participates in cardiovascular regulation and spontaneous hypertension of the rat. To investigate whether an increase in central TRH activity produces hypertension we studied the effect of the preTRH overproduction induced by I.C.V. transfection with a naked eukaryotic expression plasmid vector which encodes preTRH (pCMV-TRH). Northern blot analysis and RT-PCR showed that pCMV-TRH was transcribed in vitro and in vivo. At 24, 48, and 72 hours, pCMV-TRH (100 microg) in a significant and dose-dependent manner increased 37%, 84%, and 49%, respectively, the diencephalic TRH content and SABP (42+/-3, 50+/-2, and 22+/-2 mm Hg, respectively) with respect to the vector without the preTRH cDNA insert (V[TRH(-)]) as measured by RIA and the plethysmographic method, respectively, in awake animals. In addition, using immunohistochemistry we found that the increase of TRH was produced in circumventricular areas where the tripeptide is normally located. To further analyze the specificity of these effects we studied the actions of 23-mer sense (S), antisense (AS), and 3'self-stabilized sense (Ss) and antisense (ASs) phosphorothioate oligonucleotides against the initiation codon region. Only ASs inhibited the increase of TRH content and SABP induced by pCMV-TRH treatment. In addition, pCMV-TRH-induced hypertension seems not to be mediated by central Ang II or serum TSH. To summarize, central TRH overproduction in periventricular areas induced by I.C.V. transfection produces hypertension in rats which is reversed by specific antisense treatment. This model may help in testing effective antisense oligodeoxynucleotides against other candidate genes.

Angiotensin II enhances long-term memory in the crab Chasmagnathus.

An opaque screen moving overhead provokes an escape response in the crab Chasmagnathus granulatus that habituates after a few presentations of the eliciting stimulus. Fifteen trials with a 180-s intertrial interval or 30 trials with a 90-s interval (strong training protocol) ensures long-term habituation (LTH) of the response for 24 h, whereas 10 trials (weak training protocol) fail to induce it. However, robust LTH is obtained when crabs are injected with human angiotensin (All; 50 pmol) immediately after a weak training protocol. This memory-enhancing effect of All is dose-dependent, reversible by saralasin (5 pmol), and vanishes either when the weak training protocol is reduced to only five trials, or when the peptide is given before training or 1 h after. LTH is impaired by saralasin (5 pmol) administered before or after the strong training protocol, but no amnestic effect is disclosed when the antagonist is given 1 h after. On the other hand, both All-like immunoreactivity and angiotensin-converting enzyme-like activity are described in diverse tissues of Chasmagnathus, namely, in gills and in both thoracic and supraesophageal ganglia. Results support the view that some components of the renin-angiotensin system and their influence on memory might have emerged early in evolution.