calpain and Hypertension

Arterial aging is the major contributing factor to increases in the incidence and prevalence of cardiovascular disease, due mainly to the presence of chronic, low-grade, 'sterile' arterial inflammation. Inflammatory signaling driven by the angiotensin II cascade perpetrates adverse age-associated arterial structural and functional remodeling. The aged artery is characterized by endothelial disruption, enhanced vascular smooth muscle cell (VMSC) migration and proliferation, extracellular matrix (ECM) deposition, elastin fracture, and matrix calcification/amyloidosis/glycation. Importantly, the molecular mechanisms of arterial aging are also relevant to the pathogenesis of hypertension and atherosclerosis. Age-associated arterial proinflammation is to some extent mutable, and interventions to suppress or delay it may have the potential to ameliorate or retard age-associated arterial diseases.

Topics: Aged; Aging; Angiotensin II; Animals; Antigens, Surface; Arteries; Arteritis; Atherosclerosis; Calpain; Chemokine CCL2; Endothelin-1; Humans; Hypertension; Inflammation; Inflammation Mediators; Matrix Metalloproteinase 2; Middle Aged; Milk Proteins; Muscle, Smooth, Vascular; Nitric Oxide; Reactive Oxygen Species; Receptors, CCR2; Transforming Growth Factor beta1

Decreased Mg(2+) concentration has been implicated in altered vascular reactivity, endothelial dysfunction and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Unlike our knowledge of other major cations, mechanisms regulating cellular Mg(2+) handling are poorly understood. Until recently little was known about protein transporters controlling transmembrane Mg(2+) influx. However, new research has uncovered a number of genes and proteins identified as transmembrane Mg(2+) transporters, particularly transient receptor potential melastatin (TRPM) cation channels, TRPM6 and TRPM7. Whereas TRPM6 is found primarily in epithelial cells, TRPM7 is ubiquitously expressed. Vascular TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization and migration, and is modulated by vasoactive agents, pressure, stretch and osmotic changes. Emerging evidence suggests that vascular TRPM7 function might be altered in hypertension. The present review discusses the importance of Mg(2+) in vascular biology in hypertension and focuses on transport systems, mainly TRPM7, that might play a role in the control of vascular Mg(2+) homeostasis. Elucidation of the relationship between the complex systems responsible for regulation of Mg(2+) homeostasis, the role of TRPM7 in vascular signaling, and the cardiovascular impact will be important for understanding the clinical implications of hypomagnesemia in cardiovascular disease.

Topics: Annexin A1; Calpain; Homeostasis; Humans; Hypertension; Ion Transport; Magnesium; Models, Cardiovascular; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nonmuscle Myosin Type IIA; Oxidative Stress; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Signal Transduction; TRPM Cation Channels; Vascular Resistance; Vasodilation

In recent years interest has increased concerning the characterization of the structural-functional properties and the identification of the physiological role of non-lysosomal intracellular proteinases. Among these, calpain, a calcium-dependent cysteine proteinase ubiquitously present in a variety of tissues and cells, has been most extensively investigated in terms of activation, regulatory mechanisms, specificity and biological function. This review discusses each of these points on the basis of the most recent results concerning the general characteristics of calpain activity, and its preferential site of action within the cell as related to the specific functions of the proteinase in different cell types. As with other proteinases, calpain has to be under a continuous spatial and temporal control, and the structural and functional properties of the natural calpain inhibitor, calpastatin, must also be considered. The calpain-calpastatin system is the functional proteolytic unit that governs the activity of this intracellular proteolytic system, which is tightly correlated to the control of calcium homeostasis and thereby to the biological process of transmembrane signalling.

Topics: Calpain; Cytoskeleton; Erythrocytes; Humans; Hypertension; Nervous System Physiological Phenomena; Signal Transduction

Xin-Ji-Er-Kang (XJEK) as a herbal formula of traditional Chinese medicine (TCM) has shown the protective effects on myocardial function as well as renal function in mouse models of myocardial infarction.. We investigated the effects of XJEK on cardiovascular- and renal-function in a heart failure mouse model induced by high salt (HS) and the associated mechanisms.. For the purpose of assessing the effects of XJEK on a hypertensive heart failure model, mice were fed with 8% high salt diet. XJEK was administered by oral gavage for 8 weeks. Cardiovascular function parameters, renal function associated biomarkers and XJEK's impact on renin-angiotensin-aldosterone system (RAAS) activation were assessed. To determine the underlying mechanism, the calpain1/junctophilin-2 (JP2)/sarcoplasmic reticulum Ca. Mice on HS-diet exhibited hypertensive heart failure along with progressive kidney injury. Similar to fosinopril, XJEK ameliorated hypertension, cardiovascular-and renal- dysfunction in mice of HS-diet group. XJEK inhibited HS-induced activation of RAAS and reversed the abnormal expression pattern of calpain1and JP2 protein in heart tissues. XJEK significantly improved cell viability of angiotensin II-challenged AC16 cells. Moreover, XJEK's impact on calpain1/JP2 pathway was partly diminished in AC16 cells transfected with calpastatin siRNA.. XJEK was found to exert cardiovascular- and renal protection in HS-diet induced heart failure mouse model. XJEK inhibited HS-diet induced RAAS activation by inhibiting the activity and expression of calpain1 and protected the junctional membrane complex (JMC) in cardiomyocytes.

Topics: Animals; Blood Pressure; Calpain; Drugs, Chinese Herbal; Heart Failure; Hypertension; Kidney; Membrane Proteins; Mice; Muscle Proteins; Oxidative Stress; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction

Increased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-β in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension.. Male Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-β and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels.. SBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-β levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C.. Treatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-β and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.

Topics: Animals; Calpain; Cardiomegaly; Gene Expression Regulation; Hypertension; Male; Matrix Metalloproteinase 2; Rats; Rats, Wistar; Vasodilator Agents; Ventricular Remodeling; Verapamil

Previously, we have published that pharmacological induction of oxidative stress causes anxiety-like behavior in rats and also is associated with hypertension in these animals. Here, we report that sub-chronic induction of oxidative stress via pharmacological induction leads to i) reduction in glyoxalase (GLO)-1 and glutathione reductase (GSR)-1 expression; ii) calpain mediated reduction of brain derived neurotrophic factor (BDNF) levels; iii) NFκB mediated upregulation of proinflammatory factors interleukin (IL)-6 and tumor necrosis factor (TNF)-α and elevated angiotensin (AT)-1 receptor levels in hippocampus, amygdala and locus coeruleus regions of the brain. Acute oxidative stress has opposite effects. We speculate that regulation of GLO1, GSR1, BDNF, NFκB and AT-1 receptor may contribute to anxiety-like behavior and hypertension in rats.

Topics: Analysis of Variance; Animals; Anxiety; Brain; Brain-Derived Neurotrophic Factor; Buthionine Sulfoximine; Calpain; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glutathione Reductase; Hypertension; Inflammation; Interleukin-6; Lactoylglutathione Lyase; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha; Xanthine; Xanthine Oxidase

Females of spontaneously hypertensive (SHR strain) and normotensive rats (WKY strain and Wistar) received drinking water with normal (80 mg/liter) or reduced concentration of Ca(2+)(8 mg/liter). Activity of calcium-dependent calpain protease in neurons did not differ in 18-day-old rat pups born and suckled by these animals. Our results are consistent with published data on normal metabolism of SHR rats up to the age of 30 days.

Topics: Animals; Brain; Calcium; Calcium, Dietary; Calpain; Female; Hypertension; Myocytes, Smooth Muscle; Neurons; Proteolysis; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.

Topics: Animals; Calcium; Calpain; Cattle; Caveolae; Hypertension; Immunoassay; Molecular Weight; Muscle, Smooth, Vascular; Peptide Fragments; Protein Subunits; Pulmonary Artery

Essential hypertension (EH) is a common disorder, which can increase the risk for type 2 diabetes (T2D). Calpain-10 (CAPN10) gene was the first candidate gene of T2D identified through genome-wide linkage and positional cloning, but few works have focused on the relationship of CAPN10 with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) in EH patients.. To identify the effect of UCSNP-43 and UCSNP-44 in CAPN10 gene on susceptibility to IFG/IGT, we conducted a case-control study in 961 EH patients with and without IFG/IGT among Han Chinese population. We also evaluated the impact of two SNPs on insulin sensitivity and glucose tolerance estimated through oral glucose tolerance test and renal functions by blood chemical assays.. The major findings of this study were that UCSNP-43 displayed higher G120 and AUCg. In addition, UCSNP-44 was found associated with IFG/IGT in EH patients, and associated with increased G30, G60, AUCg, Cederholm index, Scr and eGFR. The haplotype UCSNP-43-44 was detected associated with IFG/IGT susceptibility, G60, G120, I0, AUCg, Scr and eGFR by the linear regression with the adjustment for sex, age, BMI, mean blood pressures and ACEI/ARB treatment.. These findings provided some evidence that CAPN10 gene may play an important role in the pathogenesis of IFG/IGT in EH patients.

Topics: Blood Glucose; Calpain; Female; Haplotypes; Humans; Hypertension; Insulin; Kidney; Male; Middle Aged; Polymorphism, Single Nucleotide

Diabetes and atherosclerosis may share common genetic determinants. A prior study in Hispanics found association of haplotypes in the diabetes gene calpain-10 (CAPN10) with carotid artery intima-media thickness (CIMT). This study sought to replicate this association in an independent cohort.. Four CAPN10 SNPs were genotyped and haplotypes determined in 487 Hispanic Americans from 143 families ascertained via an index case with hypertension. CIMT was measured from B-mode ultrasound, and glycemic traits quantified from euglycemic clamps. Association of SNPs and haplotypes with CIMT was determined.. The minor alleles of SNP-56 and SNP-63 were associated with increased CIMT in dominant and additive models. The association of haplotype 1112 with increased CIMT was replicated. No associations with fasting insulin, insulin secretion, or insulin sensitivity were observed.. CAPN10 association with CIMT was replicated, further supporting its role as a common genetic determinant of diabetes and atherosclerosis in Hispanics.

Topics: Adult; Calpain; Carotid Arteries; Cohort Studies; Diabetes Mellitus; Female; Genotype; Haplotypes; Hispanic or Latino; Humans; Hypertension; Insulin; Male; Tunica Intima; Tunica Media; Ultrasonography

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion. In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs. In addition, the extent of NOS degradation is a function of the level of HSP90 expression. Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein. In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place. In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity. On the basis of these findings, we propose a novel role of the HSP90-NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain. The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90-NOS heterocomplex. This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS.

Topics: Animals; Aorta; Brain; Calpain; HSP90 Heat-Shock Proteins; Hypertension; Isoenzymes; Multiprotein Complexes; Nitric Oxide Synthase; Rats; Sodium, Dietary; Tissue Extracts

Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.

Topics: Animals; Blood Pressure; Brain; Calcium; Calcium-Transporting ATPases; Calpain; Diet; Down-Regulation; Homeostasis; Humans; Hypertension; Male; Rats; Rats, Inbred Strains; Sodium Chloride, Dietary

Genes implicated in common complex disorders such as obesity, type 2 diabetes mellitus (T2DM) or cardiovascular diseases are not disease specific, since clinically related disorders also share genetic components. Cysteine protease Calpain 10 (CAPN10) has been associated with T2DM, hypertension, hypercholesterolemia, increased body mass index (BMI) and polycystic ovary syndrome (PCOS), a reproductive disorder of women in which isunlin resistance seems to play a pathogenic role. The calpain 5 gene (CAPN5) encodes a protein homologue of CAPN10. CAPN5 has been previously associated with PCOS by our group. In this new study, we have analysed the association of four CAPN5 gene variants(rs948976A>G, rs4945140G>A, rs2233546C>T and rs2233549G>A) with several cardiovascular risk factors related to metabolic syndrome in general population.. Anthropometric measurements, blood pressure, insulin, glucose and lipid profiles were determined in 606 individuals randomly chosen from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille), recruited to investigate the prevalence of anthropometric and physiological parameters related to obesity and other components of the metabolic syndrome. Genotypes at the four polymorphic loci in CAPN5 gene were detected by polymerase chain reaction (PCR).. Genotype association analysis was significant for BMI (p < or = 0.041), diastolic blood pressure (p = 0.015) and HDL-cholesterol levels (p = 0.025). Different CAPN5 haplotypes were also associated with diastolic blood pressure (DBP) (0.0005 < or = p < or = 0.006) and total cholesterol levels (0.001 < or = p < or = 0.029). In addition, the AACA haplotype, over-represented in obese individuals, is also more frequent in individuals with metabolic syndrome defined by ATPIII criteria (p = 0.029).. As its homologue CAPN10, CAPN5 seems to influence traits related to increased risk for cardiovascular diseases. Our results also may suggest CAPN5 as a candidate gene for metabolic syndrome.

Topics: Blood Pressure; Body Mass Index; Calpain; Cholesterol, HDL; Diastole; Female; Gene Frequency; Genetic Markers; Genotype; Haplotypes; Humans; Hypertension; Male; Metabolic Syndrome; Middle Aged; Phenotype; Polymorphism, Genetic; Quantitative Trait Loci

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.

Topics: Actomyosin; Amides; Angiotensin II; Animals; Antihypertensive Agents; Biomarkers; Calpain; Cardiomegaly; Cell Nucleus Division; Cullin Proteins; Cytokinesis; Heart; Hypertension; Intracellular Signaling Peptides and Proteins; Mice; Myocytes, Cardiac; Myofibrils; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Inbred Dahl; rho GTP-Binding Proteins; rho-Associated Kinases; Up-Regulation

Calpain-10 (CAPN10) has been identified as a susceptibility gene in type 2 diabetes mellitus (T2DM) and insulin resistance. The present study aimed to identify the effects of genetic variations in the CAPN10 gene on the development of type 2 diabetes and hypertension in northern Han Chinese population.. We performed a case-control study and genotyped single nucleotide polymorphism (SNP)-44, -43, -19 and -63 of CAPN10 gene in 1046 subjects from the northern China, including 493 patients with T2DM and hypertension and 553 age- and gender-matched normal healthy controls.. Univariate analysis showed that the four polymorphisms were not independently associated with T2DM and hypertension. However, the frequency distributions of SNP-44 allele C (allele 2) (17.89% vs 9.80%, P = 0.0016) and genotype CC (22) (4.21% vs 1.01%, P = 0.0059) in obese patients (body mass index > or = 30 kg/m2) were different from those in non-obese patients. Logistic regression analyses revealed that carriers of the 1112/1221 diplotype had a significantly lower odds ratio for diabetes and hypertension (OR = 0.399, 95% CI, 0.196 - 0.814, P = 0.0115). The 1112/1121 diplotype associated with significantly increased risk of type 2 diabetes in Mexican-American was not associated with the increased risk in Chinese.. These results suggested that CAPN10 gene variations might play roles in the risk of diabetes and hypertension in northern Han Chinese population.

Topics: Adult; Aged; Calpain; China; Diabetes Mellitus, Type 2; Female; Genetic Predisposition to Disease; Haplotypes; Humans; Hypertension; Linkage Disequilibrium; Male; Middle Aged; Polymorphism, Single Nucleotide; Quantitative Trait Loci

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age. The aim of the present study was to investigate the role of CALPAIN-5 (CAPN5) gene in PCOS susceptibility.. We analysed four intronic polymorphisms of the CAPN5 gene in 148 well-characterized women with PCOS and 606 unrelated controls. We performed a case-control study and an intracohort analysis of clinical characteristics associated with PCOS.. Analysis of haplotypes distribution between PCOS population compared to controls showed a strong deviation (P = 0.00029). The haplotypes GGCA and GGTG were overrepresented in PCOS patients (P = 0.009 and P = 0.001, respectively). In addition, we identified several CAPN5 haplotypes associated with phenotypic differences observed between PCOS patients, such as the presence of obesity (P = 0.02), cardiovascular complications (P = 0.02), familial antecedents of obesity (P = 0.003) and of hypertension (P = 0.007) and type 2 diabetes mellitus aggregation (P = 0.04).. These results suggest a role of CAPN5 gene in PCOS susceptibility in humans. Moreover, novel candidate risk alleles have been identified, within CAPN5 gene, which could be associated with important phenotypic and prognosis differences observed in PCOS patients.

Topics: Alleles; Calpain; Diabetes Mellitus, Type 2; Female; Gene Frequency; Genetic Predisposition to Disease; Haplotypes; Humans; Hypertension; Obesity; Phenotype; Polycystic Ovary Syndrome; Polymorphism, Single Nucleotide; Risk Factors

Calpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of the different calpain isoforms in hypertensive target organ damage. Using real-time PCR, we found calpain 6 and 9 down-regulated by more than 50% and the endogenous calpain inhibitor calpastatin up-regulated by 225%, respectively, in the hearts of Dahl salt-sensitive rats on a high salt (4% NaCl) compared to normal salt diet. On the protein level, calpain 9 but not calpastatin was regulated in the hypertensive target organs heart and kidney. Moreover, the myocardial expression of calpain 9 protein was inversely linked to left ventricular mass (r= -0.93, p<0.01), and renal expression of calpain 9 protein correlated inversely with albuminuria (r= -0.82, p<0.05). In the aorta, there was no regulation of calpain 9 on the protein level. We conclude that differential regulation of calpain 9 may play a role in hypertensive target organ damage.

Topics: Animals; Calcium-Binding Proteins; Calpain; Down-Regulation; Gene Expression Regulation, Enzymologic; Heart Diseases; Heart Ventricles; Hypertension; Isoenzymes; Kidney Diseases; Male; Rats; Rats, Inbred Dahl; RNA, Messenger

To detect the association among calpain-10(CAPN-10) gene polymorphism, hypentension and hyperglycemia.. 378 individuals in the present study were the second generation offsprings of 187 hypentensives and 19 1 nonhypertensives. Fasting plasma glucose (FPG), insulin, triglyceride and fibrinogen were determined. The polymorphism of UCSNP-43 and UCSNP-44 of CAPN 10 gene were analysed with PCR-SSCP method.. (1)The frequency of G/G geno type of UCSNP-43 was higher in the second generation offsprings of the hypertensives than that in the nonhypertensive controls(86.6%, 75.4%, P < 0.05). (2) The frequenncy of G/G genotype of UCSNP-43 was higher in the hypertensive parents of the second generation offsprings in the hypertensive groups than that in the normotensive parent of the second generation offsprings of the nonhypertensive control groups (OR = 2.84, P = 0.01). After adjustment of age, sex and body mass index (BMI), the frequency of G/G genotype in the highest FPG-quartiles (FPG 5.42 +/- 0.1mmol/L) was much more than that in the lowest FPG quartiles (FPG 4.09 +/- 0.3 mmol/L) with OR of 3.32.. Polymorphism of UCSNP-43 in CAPN-10 gene might be one of the genetic factors contributing to hypertension and diabetes mellitus in the population in Daqing city. It may be a predictor of type 2 diabetes mellitus (T2DM) in the decendents of hypertensives.

Topics: Adult; Age Factors; Blood Glucose; Calpain; Female; Fibrinogen; Gene Frequency; Genetic Diseases, Inborn; Humans; Hyperglycemia; Hypertension; Insulin; Male; Polymorphism, Genetic; Sex Factors; Triglycerides

It has been reported that the equilibrium between the erythrocyte protease calpain I and its physiological inhibitor calpastatin is disrupted in patients with essential hypertension.. To investigate the activity of non-purified calpain I in hemolysates against the erythrocytic membrane proteins, rather than against other substrates.. Evaluation of calpain I red cell activity upon its own physiological substrates in hypertensive patients, in a near-physiological environment.. LIM-23 and LIM-40 of Hospital das Clinicas of the Faculty of Medicine of USP.. Patients with moderate primary hypertension over 21 years of age who were given amlodipine (n:10) and captopril (n:10) for 8 weeks, plus normal controls (n:10).. Red cell membrane proteins were incubated with and without protease inhibitors and with and without calcium chloride and underwent polyacrylamide gel electrophoresis.. Digestion of bands 2.1 and 4.1 was observed, indicating calpain I activity. No statistical differences regarding bands 2.1 and 4.1 were observed before treatment, between the controls and the hypertensive patients, either in ghosts prepared without calcium or with increasing concentrations of calcium. Nor were statistical differences observed after treatment, between the controls and the patients treated with amlodipine and captopril, or between the patients before and after treatment with both drugs.. The final activity of non-purified calpain I upon its own physiological substrate, which was the approach utilized in this study, may more adequately reflect what happens in red cells. Under such conditions no imbalance favoring calpain I activity increase was observed. The protective factor provided by calpastatin against calpain I activity may diminish under hypertension.

Topics: Adult; Amlodipine; Ankyrins; Calcium-Binding Proteins; Calpain; Captopril; Case-Control Studies; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Hypertension; Membrane Proteins

In essential hypertension (EHT) the presence of a metabolic syndrome increases the risk of cardiovascular disease. A cell membrane abnormality is implicated but its role in cardiovascular disease is unclear. Neutrophil accumulation, which occurs by beta2-integrin (CD11b/CD18) expression, followed by release of proinflammatory factors from primary vesicles is an important factor in vascular damage. CD11b and CD69 expression on neutrophils from normal controls and EHT patients was determined by fluorescence-activated cell scanning. Neutrophils were activated with phorbol myristate acetate (PMA). Protein kinase C (PKC) and calpain were inhibited with bisindolylmaleimide and E64d, respectively. In EHT patients CD11b was not increased on neutrophils at rest. However, EHT neutrophils more readily expressed CD11b on incubation in phosphate-buffered saline and more cells went on to exocytose primary granules indicated by expression of CD69. Stimulation with PMA caused more rapid activation in EHT neutrophils with expression of CD11b, followed rapidly by exocytosis of primary granules. Bisindolylmaleimide slowed but did not prevent CD11b expression, which, together with primary granule exocytosis, continued to be faster in EHT neutrophils. E64d also slowed but did not prevent either CD11b expression or primary granule exocytosis, but this inhibitor did abolish the difference between NC and EHT neutrophils. The membrane abnormality in EHT may contribute to cardiovascular risk by increasing the rate of vesicle fusion with the cell membrane to increase neutrophil accumulation and release of inflammatory agents at sites of vascular damage. Calpain activation may be the rate-limiting component that is abnormal.

Topics: Adult; Albuminuria; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Blood Vessels; Calpain; Carcinogens; Cell Fusion; Cell Membrane; Cysteine Proteinase Inhibitors; Cytoplasmic Granules; Enzyme Inhibitors; Female; Humans; Hypertension; Indoles; Lectins, C-Type; Leucine; Lipids; Macrophage-1 Antigen; Male; Maleimides; Metabolic Syndrome; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Tetradecanoylphorbol Acetate

Calpains are cytoplasmic proteases widely distributed among eucaryotic cells. Low levels of calpain activity were found in hypertrophic hearts from hypertensive rats, but its role in hypertrophic hearts from human hypertensives is unknown. Therefore, calpain activity was investigated in erythrocytes from essential hypertensive patients in relation to their left ventricular mass.. To study the role of calpain activity in the development of left ventricular hypertrophy (LVH) in human essential hypertension.. A total of 115 hypertensives (72 untreated and 43 with treatment interrupted for at least 4 months) were included in the study. Calpain I activity was measured in human erythrocytes and LVH was measured as left ventricular mass index (LVMI) by M-mode echocardiography.. Values are given as mean+/-SEM. The hypertensives (97 men and 18 women) were 43.5+/-0.9 years old with mild to moderate levels of hypertension (systolic/diastolic blood pressure of 147.9+/-1.4/98.7+/-0.9 mmHg) and relatively recent LVH onset (3.5+/-0.5 years). An inverse relation between LVMI and erythrocytic calpain activity was present in all (P = 0.0023, R2 = 7.9%). This relation was still present considering only untreated hypertensives (P = 0.008; R2 = 9.7%), but was lost in the 43 previously treated hypertensives. Moreover, in the untreated hypertensives, after excluding the possible confounding effects of sex, age, body mass index, blood pressure and duration of hypertension, a stepwise regression showed that only two variables remained significantly related to LVMI: calpain (F = 6.23) and mean arterial pressure (F = 4.689). No relations were found between LVMI and calpastatin activity either in the whole population, or in treated or untreated hypertensives.. If we assume that the level of erythrocyte calpain activity mirrors the level in cardiomyocytes, these data seem to suggest that increased protein degradation by calpain may prevent the development of LVH in hypertensive patients. This effect is independent of the duration and severity of hypertension.

Topics: Adult; Biomarkers; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Calpain; Chromatography, Ion Exchange; Echocardiography; Erythrocytes; Female; Heart Ventricles; Humans; Hypertension; Hypertrophy, Left Ventricular; Male

We studied the activity of plasma membrane (Ca+Mg)ATPase from erythrocytes of Milan hypertensive rat strain (MHS) and Milan low calpastatin rat strain (MLCS), that show an activity level of the specific calpain inhibitor, calpastatin, about five fold reduced in comparison with the Milan normotensive rat strain (MNS), while the protease activity level is similar. This imbalance of calpain:calpastatin ratio leads to a decrease of the erythrocyte plasma membrane (Ca+Mg)ATPase activity and to the appearance of 124 kDa fragments, which are the typical products of proteolytic calpain action on the 136 kDa (Ca+Mg)ATPase native form.

Topics: Animals; Blood Pressure; Blotting, Western; Ca(2+) Mg(2+)-ATPase; Calcium-Binding Proteins; Calpain; Enzyme Inhibitors; Erythrocyte Membrane; Hypertension; Male; Molecular Weight; Rats; Rats, Mutant Strains

The Milan hypertensive strain (MHS) of rats, in addition to having hypertension, is also characterized by a genetic deficiency in calpastatin, the endogenous inhibitor of calpain. Since this protease has been implicated in long-term potentiation (LTP), we have investigated whether induction of this form of plasticity was altered in this strain of rats as compared to control animals (Milan normotensive strain, MNS). Progressive induction of LTP by increasing numbers of high frequency trains resulted in a greater degree of potentiation measured with all inducing protocols in MHS as compared with MNS animals. This difference was not related to the hypertension, since another hypertensive strain (the SHR strain) and a segregated Milan hypertensive strain, expressing only the hypertension but not the calpastatin deficiency (the MHNE strain), exhibited an LTP indistinguishable from control rats. Treatment of MHNE rats for 2 months with isovalerylcarnitine, a compound that increases calpain activity, also resulted in a greater amount of LTP induced by high frequency trains. These effects were not related to an enhancement of the NMDA receptor dependent component of responses to burst stimulation. These results are consistent with the idea that conditions under which calpain activation is facilitated are associated with a greater degree of synaptic potentiation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carnitine; Hippocampus; Hypertension; Long-Term Potentiation; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Wistar

1. The activities of glycolytic, fatty acid oxidation and citric acid cycle enzymes were measured in hypertensive and aging rat cardiac and skeletal muscles. 2. Lactate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase were significantly decreased in hypertensive, but not senescent, cardiac muscle. 3. Total phosphorylase activity was significantly increased in senescent, but not hypertensive, cardiac muscle. 4. In aging rat cardiac and skeletal muscles, calpain II titers increased significantly with age, but in normotensive and hypertensive muscles, the titers showed no significant difference.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Aging; Animals; Calpain; Citrates; Citric Acid; Fatty Acids; Glycolysis; Hypertension; L-Lactate Dehydrogenase; Male; Muscles; Myocardium; Oxidation-Reduction; Phosphorylases; Rats; Rats, Inbred SHR; Rats, Inbred WKY

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Erythrocyte Membrane; Hypertension; Membrane Proteins; Rats

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Erythrocytes; Hypertension; Macromolecular Substances; Molecular Weight; Muscles; Rats

We studied calpastatin activity in erythrocytes of Milan hypertensive and prehypertensive rats, in their normotensive controls, in F1 and F2 hybrids, and in two inbred strains derived from F2, one hypertensive and the other normotensive. Our results show that the decrease in calpastatin activity observed in Milan hypertensive rats was not caused by hypertension, it was transmitted in a recessive way in heterozygous, and it was not correlated to hypertension.

Topics: Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Erythrocytes; Heterozygote; Homozygote; Hypertension; Rats; Rats, Inbred Strains

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Topics: Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Erythrocytes; Humans; Hypertension; Kinetics; Rats

Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca2+, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca2(+)-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca2(+)-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca2(+)-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.

Topics: Anemia, Sickle Cell; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Humans; Hypertension; In Vitro Techniques; Kwashiorkor

This study examined structural and functional changes of mesenteric resistance vessels in early, developing, and established stages of hypertension development in spontaneously hypertensive rats in an attempt to identify possible mechanisms of the development and maintenance of hypertension. Our results suggest that the development of hypertension in spontaneously hypertensive rats may be caused by genetic structural and functional abnormalities of resistance vessels. Both abnormalities may be caused by hyperreactivity to norepinephrine through an altered signal transduction process, including the regulation of protein kinase C in smooth muscle cells of resistance vessels in spontaneously hypertensive rats.

Topics: Animals; Blood Pressure; Calpain; Hypertension; In Vitro Techniques; Male; Mesenteric Arteries; Models, Cardiovascular; Norepinephrine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction; Vascular Resistance

The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state.

Topics: Anion Transport Proteins; Calpain; Carrier Proteins; Chromatography, Ion Exchange; Erythrocytes; Humans; Hypertension; Phosphorylation; Protein Kinases

The calpain-calpain inhibitor system was evaluated in erythrocytes of patients with essential hypertension and normotensive controls, either with or without a family history of hypertension. Calpain levels were similar in the controls and hypertensive patients, whereas the inhibitor activity level was significantly reduced in the latter (301.8 +/- 26.4 vs 220 +/- 14 U/mg hemoglobin, p less than 0.001). Borderline hypertensive patients and a few controls with a history of hypertension showed low inhibitor activity. Similar results have recently been reported in genetically hypertensive rats of the Milan strain. A significant inverse correlation (r = -0.43, p less than 0.001) was found between mean arterial pressure and calpain inhibitor. Although the pathophysiological significance of these observations is not yet clear, they suggest a new area of investigation into the molecular mechanisms underlying essential hypertension and its complications.

Topics: Adult; Blood Pressure; Calpain; Erythrocytes; Glycoproteins; Humans; Hypertension

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.

Topics: Antibodies, Monoclonal; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytosol; Erythrocyte Aging; Erythrocyte Membrane; Erythrocytes; Humans; Hypertension; Protease Inhibitors

Rat kidney contains two different calpain isozymes distinguishable on the basis of their Ca2+ requirement and of their activation mechanisms. The two calpain isozymes are present in comparable amounts in kidney of normotensive and hypertensive rats of the Milan strain. Conversely, the level of the natural inhibitor of calpain is significantly decreased in kidney of hypertensive rats as compared to control normotensive rats. This deficiency is more pronounced in the cortical region than in other kidney fractions. These results taken together with previous observations indicating the existence of an identical defect in red cells from the same hypertensive rat strain, (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Viotti, P., Michetti, M., Duzzi, L., and Bianchi, G. (1986) Biochem. Biophys. Res. Commun. 138, 1370-1375) emphasize the possible role of an unbalanced intracellular proteolytic system in the development of genetically determined hypertension.

Topics: Animals; Calpain; Glycoproteins; Hypertension; Isoenzymes; Kidney; Kidney Cortex; Kidney Medulla; Male; Protease Inhibitors; Rats; Rats, Inbred Strains

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.

Topics: Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Calpain; Erythrocyte Membrane; Hemolysis; Humans; Hypertension; Kinetics; Phosphorylation; Reference Values

In mature red cells of rats from Milan Normal (MNS) and Hypertensive Strains (MHS), the soluble Ca2+-dependent neutral proteinase (calpain) is present in similar amounts as the form requiring 0.1-0.2 mM Ca2+ for maximum catalytic activity. The amount of the endogenous calpain inhibitor, however, differs greatly in the red cells of the two strains. In red cells from hypertensive rats the activity of the inhibitor is 10 times less with a ratio of inhibitor to calpain activity (unit/unit) of 0.2; compared to red cells from normal rats, in which this ratio is approximately 2. This is the first demonstration of the existence, in a mammalian cell, of such a low ratio of calpain to inhibitor and implies the occurrence of a potentially "unregulated" intracellular soluble proteinase. This abnormal condition may be responsible for some of the structural and metabolic changes reported in rats of the genetically determined MHS strain.

Topics: Animals; Calpain; Erythrocytes; Glycoproteins; Hypertension; Protease Inhibitors; Rats; Rats, Mutant Strains

In mature red cells of rats from Milan normal (MNS) and hypertensive strains (MHS), the soluble Ca2+ dependent neutral proteinase (calpain) is present in similar amounts with identical Mr of 110 kDa and a dimeric structure composed of two unequal subunits of Mr of 84 and 26 kDa. Conversely, the amount of the endogenous inhibitor is now confirmed by analysis of the specific activity to be approximately 10 times less in red cells of MHS rats. The inhibitor is present in red cells of both strains in three different oligomeric forms of Mr of 240, 120 and 64 kDa. This last molecular species corresponds to the single basic constituent subunit which is the reacting inhibitor form. The apparent equilibrium between the three oligomeric structures is Ca2+-dependent. The high (0.1 mM) Ca2+ requirement for the activity of calpain from erythrocytes of both strains is reduced to 1-5 microM in the presence of plasma membrane phospholipids. Activation of the enzyme in these conditions is prevented by the natural inhibitor. These results strongly support and further emphasize the hypothesis that the structural and functional abnormalities in MHS rats red cells result from an impairment in the modulation of intracellular calpain activity by interaction with its endogenous inhibitor.

Topics: Animals; Calcium; Calpain; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Erythrocytes; Hypertension; Molecular Weight; Phospholipids; Rats