calpastatin and Hypertension

Sex differences are observed in many aspects of mammalian cardiovascular function and pathology. Hypertension is more common in men than in women of the same age. Although the effects of gonadal hormones on blood pressure are considered contributing factors, the reasons for sex differences in hypertension are still not fully understood. The present study was undertaken to compare the differences in several environmental and genetic factors between men and women in the Hei Yi Zhuang, an isolated subgroup of the Zhuang minority in China.. Information on demography, diet, and lifestyle was collected in 835 women and 834 men aged 15 to 84 years. Genotyping of angiotensin-converting enzyme, adrenergic receptor beta(3), aldehyde dehydrogenase 2, calpastatin, connexin 37, hepatic lipase, lipoprotein lipase, peroxisome proliferator-activated receptor gamma, thyrotropin-releasing hormone receptor, and von Willebrand factor also was performed in these subjects.. The levels of systolic and diastolic blood pressure, and the prevalence, awareness, and treatment of hypertension were lower in women than in men (P < .05). Hypertension was positively associated with age, physical activity, alcohol consumption, body mass index, waist circumference, hyperlipidemia, total energy, total fat, sodium intake, and sodium/potassium ratio, and negatively associated with education level, total dietary fiber, potassium intake, angiotensin-converting enzyme, aldehyde dehydrogenase 2, and hepatic lipase genotypes in men (P < .05). Hypertension was positively associated with age, hyperlipidemia, total energy, total fat, sodium intake, sodium/potassium ratio, calpastatin, and von Willebrand factor genotypes, and negatively associated with education level, total dietary fiber, potassium, calcium intake, lipoprotein lipase, and thyrotropin-releasing hormone receptor genotypes in women (P < .05).. Sex differences in the prevalence of hypertension in the Hei Yi Zhuang population may be mainly attributed to the differences in dietary habits, lifestyle choices, sodium and potassium intakes, physical activity level, and some genetic polymorphisms.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Calcium-Binding Proteins; China; Connexins; Female; Gap Junction alpha-4 Protein; Genotype; Humans; Hypertension; Life Style; Lipase; Lipoprotein Lipase; Male; Middle Aged; Obesity; Peptidyl-Dipeptidase A; Polymorphism, Genetic; PPAR gamma; Prevalence; Receptors, Adrenergic, beta-3; Receptors, Thyrotropin-Releasing Hormone; Risk Factors; Sex Factors; von Willebrand Factor

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

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

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

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.

Topics: Age Factors; Aging; Animals; Brain; Calcium-Binding Proteins; Chromatography, Ion Exchange; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hypertension; Immunoblotting; Kidney; Models, Biological; Myocardium; Rats; RNA, Messenger; Tissue Distribution

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

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

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