Low Intracellular Calcium Levels Research Articles

Parkinson Disease Protein 7 (PARK7) Is Related to the Ability of Mammalian Sperm to Undergo In Vitro Capacitation.

Parkinson disease protein 7 (PARK7) is a multifunctional protein known to be involved in the regulation of sperm motility, mitochondrial function, and oxidative stress response in mammalian sperm. While ROS generation is needed to activate the downstream signaling pathways required for sperm to undergo capacitation, oxidative stress has detrimental effects for sperm cells and a precise balance between ROS levels and antioxidant activity is needed. Considering the putative antioxidant role of PARK7, the present work sought to determine whether this protein is related to the sperm ability to withstand in vitro capacitation. To this end, and using the pig as a model, semen samples were incubated in capacitation medium for 300 min; the acrosomal exocytosis was triggered by the addition of progesterone after 240 min of incubation. At each relevant time point (0, 120, 240, 250, and 300 min), sperm motility, acrosome and plasma membrane integrity, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium and ROS were evaluated. In addition, localization and protein levels of PARK7 were also assessed through immunofluorescence and immunoblotting. Based on the relative content of PARK7, two groups of samples were set. As early as 120 min of incubation, sperm samples with larger PARK7 content showed higher percentages of viable and acrosome-intact sperm, lipid disorder and superoxide levels, and lower intracellular calcium levels when compared to sperm samples with lower PARK7. These data suggest that PARK7 could play a role in preventing sperm from undergoing premature capacitation, maintaining sperm viability and providing a better ability to keep ROS homeostasis, which is needed to elicit sperm capacitation. Further studies are required to elucidate the antioxidant properties of PARK7 during in vitro capacitation and acrosomal exocytosis of mammalian sperm, and the relationship between PARK7 and sperm motility.

Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry.

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis.

BackgroundPreviously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling.MethodsIn vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo.FindingsCACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ).InterpretationCACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling.FundingCanadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.

Cryotolerance of Stallion Spermatozoa Relies on Aquaglyceroporins rather than Orthodox Aquaporins.

Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen–thawed samples. In the presence of AC, different parameters were altered (p < 0.05), but not in a concentration- or time-depending manner. PHL was found to decrease sperm motility, viability, acrosome integrity, and the percentages of spermatozoa with low membrane lipid disorder, high mitochondrial membrane potential (MMP) and high intracellular levels of calcium and superoxides (p < 0.05). Finally, the sperm motility, viability, acrosome integrity, the percentages of spermatozoa with low membrane lipid disorder, high MMP and high intracellular calcium levels were higher (p < 0.05) in PDO treatments than in the control. The sperm response to AC, PHL and PDO indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Furthermore, post-thaw sperm quality was higher in PDO treatments than in the control, suggesting that this molecule is a potential permeable cryoprotectant.

Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. We were able by using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant) to stabilize H460 apoptotic cells in cell cultures for at least 72hours preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cells characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels, plasma membrane potential, PS externalization and ability of being phagocytosed. Stabilized apoptotic cells can be considered as dying cells in which the cellular cortex and plasma membrane are maintained intact or alive. In a metaphorical sense, we can consider them as "living dead" or "zombie cells". Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

Bisphosphonate Therapy of Osteoporosis Predisposes Epithelial Breakdown in Osteonecrosis of the Jaws, with Calcium Ion Constraint

Osteoporosis is a common bone disease prevalent in aging in humans deriving from all ethnic backgrounds. Bisphosphonates (BPs) influence the metabolism of soft tissues, including direct toxicity on oral mucosae. This contributes to mucosal ulceration and exposure of underlying bone (1) The ulcerative necrosis is prevalent on sites when thin mucosa covers bony protuberances, prominences or exostoses. BONJ cases are often preceded by dento-alveolar therapy. Dental treatment procedures, like deep root-planing, extractions and surgical placement of osseo- integrated implants are acknowledged as predisposing practices which precipitate BONJ. The increased demand for calcium ions necessary for bone regeneration and repair, exceeds the dysfunctional osseous reserve, and results in epithelial break down and localized osseous necrosis. Oral biofilm is highly infectious, and without intact epithelium and resilient bone, BONJ develops. Consequently because oral surgical procedures are central in developing BONJ, not only should all dentists must be aware of this and be ultra-vigilant about all patients being treated with bisphosphonates, but all dental treatments should be concluded before starting the bisphosphonate therapy. When the normally very low (10-8 M) intra-cellular calcium levels rise, the gap junctions undergo conformation changes that close the gap junctions channel, inhibiting transfer of other metabolites (2).

Active vitamin D deficiency mediated by extracellular calcium and phosphorus results in male infertility in young mice.

We used mice with targeted deletion of 25-hydroxyvitamin D-1 α-hydroxylase [1α(OH)ase(-/-)] to investigate whether 1,25(OH)2D3 deficiency results in male infertility mediated by 1,25(OH)2D3 or extracellular calcium and phosphorus. Male 1α(OH)ase(-/-) and their wild-type littermates fed either a normal diet or a rescue diet from weaning were mated at 6-14 wk of age with female wild-type mice on the same diet. The fertility efficiency of females was analyzed, and the reproductive phenotypes of males were evaluated by histopathological and molecular techniques. Hypocalcemic and hypophosphatemic male 1α(OH)ase(-/-) mice on a normal diet developed infertility characterized by hypergonadotropic hypogonadism, with downregulation of testicular calcium channels, lower intracellular calcium levels, decreased sperm count and motility, and histological abnormalities of the testes. The proliferation of spermatogenic cells was decreased with downregulation of cyclin E and CDK2 and upregulation of p53 and p21 expression, whereas apoptosis of spermatogenic cells was increased with upregulation of Bax and p-caspase 3 expression and downregulation of Bcl-xl expression. When serum calcium and phosphorus were normalized by the rescue diet, the defective reproductive phenotype in the male 1α(OH)ase(-/-) mice, including the hypergonadotropic hypogonadism, decreased sperm count and motility, histological abnormalities of testis, and defective spermatogenesis, was reversed. These results indicate that the infertility seen in male 1,25(OH)2D3-deficient mice is not a direct effect of active vitamin D deficiency on the reproductive system but is an indirect effect mediated by extracellular calcium and phosphorus.

Stabilization of apoptotic cells: generation of zombie cells.

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.

L-type calcium channel modulates cystic kidney phenotype

In polycystic kidney disease (PKD), abnormal proliferation and genomic instability of renal epithelia have been associated with cyst formation and kidney enlargement. We recently showed that L-type calcium channel (CaV1.2) is localized to primary cilia of epithelial cells. Previous studies have also shown that low intracellular calcium level was associated with the hyperproliferation phenotype in the epithelial cells. However, the relationship between calcium channel and cystic kidney phenotype is largely unknown. In this study, we generated cells with somatic deficient Pkd1 or Pkd2 to examine ciliary CaV1.2 function via lentiviral knockdown or pharmacological verapamil inhibition. Although inhibition of CaV1.2 expression or function did not change division and growth patterns in wild-type epithelium, it led to hyperproliferation and polyploidy in mutant cells. Lack of CaV1.2 in Pkd mutant cells also decreased the intracellular calcium level. This contributed to a decrease in CaM kinase activity, which played a significant role in regulating Akt and Erk signaling pathways. Consistent with our in vitro results, CaV1.2 knockdown in zebrafish and Pkd1 heterozygous mice facilitated the formation of kidney cysts. Larger cysts were developed faster in Pkd1 heterozygous mice with CaV1.2 knockdown. Overall, our findings emphasized the importance of CaV1.2 expression in kidneys with somatic Pkd mutation. We further suggest that CaV1.2 could serve as a modifier gene to cystic kidney phenotype.

Snapin accelerates exocytosis at low intracellular calcium concentration in mouse chromaffin cells

Snapin associates with SNAP-25 and with assembled SNARE complexes, stabilizing the coupling between Synaptotagmin-1 and SNAP-25. Deletion of Snapin reduces releasable pools of vesicles in chromaffin cells and reduces synchronous release of neurotransmitter in cortical neurons. Snapin deletion leads to a deficit in exocytosis at low calcium concentration with no change in the threshold calcium concentration for exocytosis in chromaffin cells. In order to determine whether Snapin deletion alters release rates or calcium dependence, we examined the effect of overexpression of wild type Snapin on readily releasable pool kinetics and pool size in mouse chromaffin cells. Modest increases in intracellular calcium induced by flash-photolysis unmasked a rapidly releasing component of secretion which was enhanced when Snapin was overexpressed. This result indicates that Snapin allows rapid release at lower intracellular calcium levels at which release of the remaining RRP occurs more slowly.

N-methyl-d-aspartate receptor subunit NR3a expression and function in principal cells of the collecting duct

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP3) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP3. This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP3. The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.

Control of Antigen Presentation with a Photoreleasable Agonist Peptide

The immunological synapse is a specialized intercellular junction between a T cell and a target cell that orchestrates the engagement of receptors and ligands in space and time as a means of regulating function. Here we introduce a reagent for controlling the spatial and temporal presentation of natural antigen to T cells. Moth cytochrome c (88-103) peptide (MCC), an agonist to the murine T cell receptor AND when presented in the context of H2 IEk major histocompatibility complex (IEk), was synthesized with the side-chain amine of Lys99 conjugated to a photosensitive protecting group, 6-nitroveratryloxycarbonyl (NVOC). Cells plated on supported bilayers displaying mobile intercellular adhesion molecule-1 (ICAM-1) and NVOC-MCC loaded IEk did not form immunological synapses and exhibited low intracellular calcium levels, similar to cells presented with self-peptide. Irradiation with UV light was sufficient to restore agonist activity in situ.

Darier disease--novel mutations in ATP2A2 and genotype-phenotype correlation.

Darier disease (DD) is with a frequency of up to 1 in 36,000 a relatively common genodermatosis with autosomal dominant inheritance and late age of onset. The progressive skin manifestations are variable, but often debilitating and disfiguring, and may be associated with a wide range of neuropsychiatric problems, such as epilepsy and depression. On histology, acantholysis and dyskeratosis are prominent findings, implicating impaired functionality of desmosomes. Recently, mutations in the ATP2A2 gene encoding SERCA2, a calcium pump of the endo/sacrcoplasmic reticulum, have been identified as the molecular basis of DD. This slow-twitched calcium ATPase has two splice variants, one of which is highly expressed in epidermis, and maintains low intracellular calcium levels by facilitating transport of cytosolic calcium into the endoplasmic reticulum. Thus, it may confer a direct effect on the established calcium-dependent assembly of desmosomes. We screened ATP2A2 in a cohort of 24 DD families using conformation sensitive gel electrophoresis and direct sequencing, and detected 14 distinct mutations, 9 of which were novel. The mutational spectrum included 9 missense mutations, 1 nonsense mutation, 3 small in-frame deletions, and a 19-basepair insertion. Mutations were scattered over the entire gene with a slight preponderance in the first 8 exons, and affected exclusively residues conserved among all SERCAs. In addition, we found 2 silent polymorphisms, 1 of which occurred in 4 unrelated families. Comparison of molecular data and phenotypic features, such as severity and type of disease, occurrence of mucosal involvement, or association with neuropsychiatric disorders, did not reveal an obvious genotype-phenotype correlation in our cohort.

Cellular physiology of the neonatal rat cerebral cortex: intrinsic membrane properties, sodium and calcium currents.

The cellular physiology of the primary somatosensory cortex was studied in postnatal day (P) 0 to P5 rats using whole-cell patch-clamp recordings. Visually identified Cajal-Retzius, subplate, bifurcated pyramidal, and immature, putatively migrating neurons showed resting membrane potentials between -44 and -50 mV and TTX-sensitive action potentials. Immature pyramidal neurons with the smallest surface area ( approximately 1,600 microm(2)) revealed the largest input resistance ( approximately 1.8 GOmega), and subplate cells with the largest surface area ( approximately 6,200 microm(2)) showed an input resistance of approximately 1 GOmega. Ontogenetically older Cajal-Retzius and subplate cells revealed shorter and larger action potentials compared to bifurcated and immature pyramidal neurons. Whereas Cajal-Retzius and subplate cells responded to injection of depolarizing current pulses with a repetitive nonadapting and fast spiking firing pattern, immature pyramidal neurons showed strong adaptation. Subplate cells revealed the fastest action potentials, largest sodium current amplitude (-714 pA), and highest sodium current density (-38 microA/cm(2)), enabling these cells to transmit afferent activity faithfully to postsynaptic neurons. Whereas all cell types expressed a high-voltage-activated (HVA) calcium current, none of them showed a significant low-voltage-activated calcium current. The largest peak (-25.5 microA/cm(2)) and steady-state (-7.6 microA/cm(2)) HVA calcium current density could be observed in immature presumed migrating neurons. In contrast, Cajal-Retzius and subplate neurons showed a significantly lower peak (-4.9 microA/cm(2)) and steady-state (<-3.3 microA/cm(2)) HVA calcium current density. Whereas a large HVA calcium current may promote neuronal migration of immature neurons, low intracellular calcium levels may provoke apoptosis in Cajal-Retzius and subplate cells.

Protein oxidation and age-dependent alterations in calcium homeostasis.

Alterations in the capacity to maintain normal calcium homeostasis have been suggested to underlie the reduced cellular function characteristic of the aging process, and to predispose the senescent organism to a host of diverse pathologies including cancer, heart disease, and a range of muscle and neurodegenerative diseases. Therefore, critical to the eventual treatment of many age-related diseases has been the identification of both post-translational modifications and the underlying structural changes that result in an age-related decline in the function of critical calcium regulatory proteins. In brain, multiple methionines within the calcium signaling protein calmodulin (CaM) are oxidized to their corresponding methionine sulfoxides during aging, resulting in an inability to activate a range of target proteins, including the plasma membrane (PM) Ca-ATPase involved in the maintenance of the low intracellular calcium levels necessary for intracellular signaling. Likewise, changes in the transport activity of the PM-Ca-ATPase occur during aging. In muscle, the function of the SERCA2a isoform of the Ca-ATPase within the sarcoplasmic reticulum (SR) declines during aging as a result of the nitration of selected tyrosines. The age-related loss-of-function of these critical calcium regulatory proteins are consistent with observed increases in intracellular calcium levels within senescent cells. A possible regulatory role for these post-translational modifications is discussed, since they have the potential to be reversed following the restoration of normal cellular redox conditions by intracellular repair enzymes that are specific for these post-translational modifications. It is suggested that the reversible oxidation of critical calcium regulatory proteins within excitable cells by reactive oxygen species functions to enhance cellular survival under conditions of oxidative stress by reducing the energy expenditure within excitable cells. Thus, a diminished ability to efficiently generate cellular ATP may ultimately underlie the loss of calcium homeostasis and cellular function during aging.

Ordered and Cooperative Binding of Opposing Globular Domains of Calmodulin to the Plasma Membrane Ca-ATPase

We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Contact sites between the inner and outer mitochondrial membranes in stunned versus hibernating myocardium

This study characterizes the energy state and the influence of calcium on myocardial stunning and chronic hibernation via the quantification of a calcium-sensitive phenomenon known as mitochondrial contact sites. For stunning, the left anterior descending artery of mongrel dogs was occluded for 15 minutes, followed by a 150-minute reperfusion; for chronic hibernation, we used human biopsies obtained during coronary artery bypass graft (CABG) from viable (positron emission tomography-controlled) asynergic areas. Both sample groups were processed for electron microscopy, and the ratio of surface densities of contact sites to mitochondrial membranes was quantified with morphometry. Therefore a cycloidal pattern was superimposed on the electron micrographs, and the ratio between the total number of intersections between cycloids and contact sites and the total number of intersections between cycloids and mitochondrial membranes is the ratio of surface densities ( S s ). In stunned cells, S s = 0.46 ± 0.06, which is significantly higher than the ratio in the normokinetic cells, S s = 0.355 ± 0.003, although the general ultrastructure of the subcellular compartments is practically identical to those in the normoxic area. The distinction between cells affected by chronic hibernation and normal cells was based on structural criteria. The ratio of surface densities, expressed S s = 0.27 ± 0.05, was significantly lower than the ratio in the normoxic area ( S s = 0.356 ± 0.005). The high ratio of surface densities in the stunned cells lends credibility to the notion that stunning implies an increased intracellular calcium content and energy demand, whereas hibernation might be a kind of low demand-low supply situation with a low intracellular calcium level.

Calcium transport in diluted or cooled ram semen.

Ram semen was subjected to various dilution rates and temperatures of dilution, and was also subjected to slow cooling and rewarming. Calcium ion movements across sperm membranes were measured using the radioisotope 45Ca2+. It was shown that even 2- to 4-fold dilution caused an increase in intracellular calcium content. An increase in intracellular calcium also occurred in proportion to a decreased temperature of dilution. After an initial increase in intracellular calcium content, spermatozoa appeared able to restore a low intracellular calcium level over a period of 2 h at 22 degrees C or above. This ability was lost at 16 degrees C or below. However, if undiluted semen was slowly cooled (0.125 degrees C/min) even to 5 degrees C and rewarmed to 22 degrees C before dilution, the spermatozoa regained this ability. In contrast, spermatozoa rapidly cooled to 5 degrees C and rewarmed to 22 degrees C before dilution were not able to restore the low intracellular calcium level.