Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum.

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca2+ homeostasis. Therefore, the plasma membrane Ca2+-ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

High-throughput measurement of the Ca2+-dependent ATPase activity in COS microsomes.

We provide a detailed procedure to determine the Ca(2+)-dependent ATPase activity in COS or HEK293 cells overexpressing a Ca(2+) pump. The ATPase activity is determined by the Baginsky method, which allows measurement of the steady-state production of inorganic phosphate (Pi). We have adapted this widely applied method into a sensitive, fast, and semi-high-throughput protocol suitable for use in a 96-well plate format.

The Ca(2+)/H(+) antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca(2+) ATPase (SPCA1).

Plasma membrane Ca(2+)-ATPase 2 (PMCA2) knockout mice showed that ~60% of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca(2+)-ATPase's 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca(2+)/H(+) antiporter (TMEM165) is expressed sufficiently in the Golgi of lactating mammary tissue to be a relevant contributor to secretory pathway mammary calcium transport. TMEM165 shows marked expression on day one of lactation when compared to timepoints prepartum. At peak lactation TMEM165 expression was 25 times greater than that of early pregnancy. Forced cessation of lactation resulted in a rapid ~50% decline in TMEM165 expression at 24h of involution and TMEM165 expression declined 95% at 96 h involution. It is clear that the timing, magnitude of TMEM165 expression and its Golgi location supports a role for this Golgi Ca2(+)/H(+) antiporter as a contributor to mammary Golgi calcium transport needs, in addition to the better-characterized roles of SPCA1&2.

Decreases in plasma membrane Ca²âº-ATPase in brain synaptic membrane rafts from aged rats.

Precise regulation of free intracellular Ca(2+) concentrations [Ca(2+) ](i) is critical for normal neuronal function, and alterations in Ca(2+) homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca(2+) ](i) is the plasma membrane Ca(2+) -ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca(2+) . We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.

Effects of cadmium on calcium transporter SPCA, calcium homeostasis and ß-casein expression in the murine mammary epithelium.

Maternal cadmium (Cd) exposure during lactation causes neurobehavioral effects in the suckling offspring as well as involution like disturbances in the mammary glands of rodents. The aim of the present study was to examine Cd-induced effects in secreting mammary epithelial cells in relation to calcium (Ca) transport and ß-casein expression. Reduced protein expression of secretory pathway Ca-ATPase (SPCA) was revealed in the mammary glands of lactating mice exposed to Cd during peak lactation. In concordance, SPCA gene expression was down regulated and total intracellular Ca levels reduced in murine mammary epithelial HC11 cells treated with Cd for 72 h. Cd reduced ß-casein gene expression in a concentration dependent manner in the HC11 cells. Our findings on Cd-induced reduction of Ca levels, SPCA and ß-casein expression in the mammary epithelium resemble the effects observed in the mammary glands as a result of forced weaning. In conclusion, maternal Cd exposure during lactation may disturb Ca regulation and decrease the levels of ß-casein in milk with potential nutritional and developmental implications for the breast-fed newborn.

MIST1 regulates the pancreatic acinar cell expression of Atp2c2, the gene encoding secretory pathway calcium ATPase 2.

MIST1 is a transcription factor expressed in pancreatic acinar cells and other serous exocrine cells. Mice harboring a targeted deletion of the Mist1 gene (Mist1(-/-)) exhibit alterations in acinar regulated exocytosis and aberrant Ca(2+) signaling that are normally controlled by acinar cell Ca(2+)-ATPases. Previous studies indicated that total sarcoendoplasmic reticulum Ca(2+)-ATPases (SERCA) and plasma membrane Ca(2+)-ATPases (PMCA) remained unaffected in Mist1(-/-) acinar cultures. Therefore, we have assessed the expression of Atp2c2, the gene that encodes the secretory pathway Ca(2+)-ATPase 2 (SPCA2). We revealed a dramatic decrease in pancreatic expression of Atp2a2 mRNA and SPCA2 protein in Mist1(-/-) mice. Surprisingly, this analysis indicated that the acinar-specific Atp2c2 mRNA is a novel transcript, consisting of only the 3' end of the gene and the protein and localizes to the endoplasmic reticulum. Expression of SPCA2 was also lost in Mist1(-/-) secretory cells of the salivary glands and seminal vesicles, suggesting that Atp2c2 transcription is regulated by MIST1. Indeed, inducible MIST1 expression in Mist1(-/-) pancreatic acinar cells restored normal Atp2c2 expression, supporting a role for MIST1 in regulating the Atp2c2 gene. Based on these results, we have identified a new Atp2c2 transcript, the loss of which may be linked to the Mist1(-/-) phenotype.

Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase.

Ca2+-ATPase cytochemistry frequently uses the incubation medium of Ando et al. that was introduced in 1981. Some studies, however, have suggested that this medium localizes ecto-ATPase in addition to Ca2+-ATPase and that Ca2+-ATPase is sensitive to fixation. Strong activity of the enzyme on the luminal surface of the blood-brain barrier (BBB) also is considered indicative of immature or pathological microvessels. We address here five questions. 1) Is the incubation medium of Ando et al. specific for BBB Ca2+-ATPase or does it also localize ecto-ATPase? 2) How are the two enzymes distributed in the BBB? 3) How would data interpretation be prone to error if the cytochemical study does not use controls identifying ecto-ATPase? 4) Does the amount of reaction product of both enzymes vary significantly when the cortical tissue is exposed to different fixatives? 5) Does the presence of Ca2+-ATPase on the luminal membrane of the BBB necessarily indicate immature or abnormal brain endothelial cells? Adult male Sprague-Dawley rats were perfused with one of two different fixatives and vibratome slices of the brain cortex were incubated in the medium of Ando et al. The controls used were those demonstrating the ecto-ATPase and those that do not. The results indicate that the incubation medium is not specific for Ca2+-ATPase, because it also localizes the ecto-ATPase. Ca2+-ATPase appears to be localized primarily on the luminal surface of the BBB, while ecto-ATPase is localized on both the luminal and abluminal surfaces. The portion of the reaction product contributed by Ca2+-ATPase would not have been identified if the controls uniquely identifying the ecto-ATPase had not been used. The amount of reaction product formed by Ca2+-ATPase is strongly dependent on the type of fixative used. The strong localization of Ca2+-ATPase on the luminal surface of the BBB is not only normal, but also better accounts for the physiological homeostasis of Ca2+ across the blood-brain interface and should not be interpreted as indicative of immature or pathological microvessels.

Differential cell-specific location of Cav-1 and Ca(2+)-ATPase in terminal Schwann cells and mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor.

Caveolae are involved in clathrin-independent endocytosis, transcytosis, signal transduction, and tumor suppression - all of which depend on their main constituent protein caveolin families. The periodontal Ruffini ending has been reported to develop a caveola-like structure on the cell membrane of both the axon terminals and Schwann sheaths, suggesting the existence of an axon-Schwann cell interaction in the periodontal Ruffini endings. However, little information is available concerning the functional significance of these caveolae. The present study was undertaken to examine the immunolocalization of caveolin-1, -3 (Cav-1, Cav-3) and Ca(2+)-ATPase in the periodontal Ruffini endings of the rat incisor. Decalcified sections of the upper jaws were processed for immunocytochemistry at the levels of light and electron microscopy. Some immunostained sections were treated with histochemistry for nonspecific cholinesterase (nChE) activity. Observations showed the periodontal Ruffini endings were immunopositive for Cav-1, but not Cav-3. Immunoreactive products for Cav-1 were confined to caveola-like structures in the cell membranes of the cytoplasmic extensions and cell bodies of the terminal Schwann cells associated with the periodontal Ruffini endings. However, the axonal membranes of the terminals did not express any Cav-1 immunoreaction. Double staining with Ca(2+)-ATPase and either protein gene product 9.5 (PGP 9.5) or S-100 protein disclosed the co-localization of immunoreactions in the axonal branches of the periodontal Ruffini endings, but not in the terminal Schwann cells. As Ca(2+) plays an important role in mechanotransduction, these characteristic immunolocalizations show Cav-1/Ca(2+)-ATPase might be involved in the quick elimination of intracellular Ca(2+) in mechanotransduction.

A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process.

Ca(2+) is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca(2+) pumps conferred the ability to grow on Ca(2+)-depleted medium and tolerance to toxic levels of Mn(2+). A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca(2+) and Mn(2+) loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca(2+), indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 microm Mn(2+), indicating that AtECA3 is also important for the detoxification of excess Mn(2+). Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca(2+)-stimulated root growth and the detoxification of high Mn(2+), possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall.

Localization of plasma membrane and secretory calcium pumps in the mammary gland.

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

[Organic features of mast cells cytochemistry in rats].

The mast cells from small intestine mesentery, brain membrane, skin, uterus, wide uterical ligament, ovaries and peritoneal fluid were studied in rats by hystochemical and immunocytochemical technique. Constitutional and inducible NO-synthase, Ca2+-ATPase, cytochromoxidase, and biogenic amines (histamine, dopamine, serotonin) were revealed in mast xells, localized in different tissues. A great number of mast cells with positive cytochromoxidase reaction was presented in skin, intestine mesentery and peritoneal fluid, while the amount of must cells containing biogenic amines was found to be greater in the organs with a significant deal of Ca2+-ATPase and NO-synthase-active cells. The number of degranulating mast cells was lower in the organs with a lack of such cells (skin, wide uterical ligament).

Yeast P4-ATPases Drs2p and Dnf1p are essential cargos of the NPFXD/Sla1p endocytic pathway.

Drs2p family P-type ATPases (P4-ATPases) are required in multiple vesicle-mediated protein transport steps and are proposed to be phospholipid translocases (flippases). The P4-ATPases Drs2p and Dnf1p cycle between the exocytic and endocytic pathways, and here we define endocytosis signals required by these proteins to maintain a steady-state localization to internal organelles. Internalization of Dnf1p from the plasma membrane uses an NPFXD endocytosis signal and its recognition by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, Sla2p/End4p, and End3p. Drs2p has multiple endocytosis signals, including two NPFXDs near the C terminus and PEST-like sequences near the N terminus that may mediate ubiquitin (Ub)-dependent endocytosis. Drs2p localizes to the trans-Golgi network in wild-type cells and accumulates on the plasma membrane when both the Ub- and NPFXD-dependent endocytic mechanisms are inactivated. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be endocytosed through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo in cells compromised for Ub-dependent endocytosis. These results demonstrate an essential role for endocytosis in retrieving proteins back to the Golgi, and they define critical cargos of the NPFXD/Sla1p system.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

BACKGROUND: Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. METHODS: Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. RESULTS: We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). CONCLUSIONS: Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Molecular determinants of altered Ca2+ handling in human chronic atrial fibrillation.

BACKGROUND: Abnormal Ca2+ handling may contribute to impaired atrial contractility and arrhythmogenesis in human chronic atrial fibrillation (cAF). Here, we assessed the phosphorylation levels of key proteins involved in altered Ca2+ handling and contractility in cAF patients. METHODS AND RESULTS: Total and phosphorylation levels of Ca2+-handling and myofilament proteins were analyzed by Western blotting in right atrial appendages of 49 patients in sinus rhythm and 52 cAF patients. We found a higher total activity of type 1 (PP1) and type 2A phosphatases in cAF, which was associated with inhomogeneous changes of protein phosphorylation in the cellular compartments, ie, lower protein kinase A (PKA) phosphorylation of myosin binding protein-C (Ser-282 site) at the thick myofilaments but preserved PKA phosphorylation of troponin I at the thin myofilaments and enhanced PKA (Ser-16 site) and Ca2+-calmodulin protein kinase (Thr-17 site) phosphorylation of phospholamban. PP1 activity at sarcoplasmic reticulum is controlled by inhibitor-1 (I-1), which blocks PP1 in its PKA-phosphorylated form only. In cAF, the ratio of Thr-35-phosphorylated to total I-1 was 10-fold higher, which suggests that the enhanced phosphorylation of phospholamban may result from a stronger PP1 inhibition by PKA-hyperphosphorylated (activated) I-1. CONCLUSIONS: Altered Ca2+ handling in cAF is associated with impaired phosphorylation of myosin binding protein-C, which may contribute to the contractile dysfunction after cardioversion. The hyperphosphorylation of phospholamban probably results from enhanced inhibition of sarcoplasmic PP1 by hyperphosphorylated I-1 and may reinforce the leakiness of ryanodine channels in cAF. Restoration of sarcoplasmic reticulum-associated PP1 function may represent a new therapeutic option for treatment of atrial fibrillation.

[Influences of some homeopathic preparations on ionic homeostasis at different dilutions].

Considerable number of the investigations are dedicated to the study of the influences of the various agents on ionic homeostasis of the cell. However, there are actually no works related to the impact on these indices of homeopathic preparations (HP). In the present work influence of HP - stimulated phosphoric acid (PA), at low dilutions (10(-14) and 10(-42)) and non-stimulated PA, at high dilutions (10(-200) and 10(-400)), on transmembrane transport of Na+, K+, Ca2+, and enzymes - Na,K-ATPase and Ca-ATPase - was investigated. Experiments were carried out by means of ion-selective electrodes, in order to measure concentration of ions in the Ringer solution. Results have shown that HP at low dilution always stimulated observed indices, while HP at high dilution - suppressed these indices. Therefore, driving force of HP increases with dilution, because number of hydrate complexes increases and so does velocity of information transmission, which is responsible for different effects.

[Influence of electromagnetic radiation of different ranges on the transmembrane transport of Na+, K+, and Ca2+ ions in normal and tumor cells].

The problem of planetary pollution is one of the most pressing ones. In the present work it was determined to assess functional state of a cell according to criterion of transport of Na(+), K(+), and Ca(2+) ions subjected to irradiation with electromagnetic fields (EMF) of different frequency ranges. Recording of the ions' concentration was made with the ion-selective electrodes immersed into the Ringer solution. The ion transport was studied in the fibroblasts of the mice. Influences of the EMF in the range of 20 to 100 Hz and different inductances were assessed. It was found that the frequencies below 45 Hz and above 60 Hz, at inductance of 2,0 mT induced stimulation of transmembrane transport of Na(+), K(+), and Ca(2+) ions. According to some electrophysiologists, this effect must be due to the fact that the frequency below 45 Hz generates potentials too slowly. Therefore an adaptation to this process does occur, while at the higher frequencies velocity of stimulation is so high that the cellular membrane cannot react and, as a result, the effect similar to the first variant is observed.

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa.

Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37 degrees Celsius for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca(2+)-ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca(2+)-ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca(2+)-ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis.

Deletion of the inducible 70-kDa heat shock protein genes in mice impairs cardiac contractile function and calcium handling associated with hypertrophy.

BACKGROUND: Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored. METHODS AND RESULTS: Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen-activated protein kinase (p38-MAPK), Raf-1, and extracellular signal-regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1-ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a. CONCLUSIONS: Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.

Altered calcium handling is an early sign of streptozotocin-induced diabetic cardiomyopathy.

The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP-ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca(2+)(i)) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca(2+)(i) concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca(2+)(i) level (control, 111-/+20 vs DM, 221-/+35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na(+)/Ca(2+) exchanger. In response to beta-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca(2+)(i) handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling.