Low Free Ca2 Research Articles

Functional activity and morphology of isolated rat cardiac mitochondria under calcium overload. Effect of naringin.

The function of mitochondria as a regulator of myocyte calcium homeostasis has been extensively discussed. The aim of the present work was further clarification of the details of modulation of the functional activity of rat cardiac mitochondria by exogenous Ca2+ ions either in the absence or in the presence of the plant flavonoid naringin. Low free Ca2+ concentrations (40-250 nM) effectively inhibited the respiratory activity of heart mitochondria, remaining unaffected the efficacy of oxygen consumption. In the presence of high exogenous Ca2+ ion concentrations (Ca2+ free was 550 µM), we observed a dramatic increase in mitochondrial heterogeneity in size and electron density, which was related to calcium-induced opening of the mitochondrial permeability transition pores (MPTP) and membrane depolarization (Ca2+free ions were from 150 to 750 µM). Naringin partially prevented Ca2+-induced cardiac mitochondrial morphological transformations (200 µM) and dose-dependently inhibited the respiratory activity of mitochondria (10-75 µM) in the absence or in the presence of calcium ions. Our data suggest that naringin (75 µM) promoted membrane potential dissipation, diminishing the potential-dependent accumulation of calcium ions by mitochondria and inhibiting calcium-induced MPTP formation. The modulating effect of the flavonoid on Ca2+-induced mitochondria alterations may be attributed to the weak-acidic nature of the flavonoid and its protonophoric/ionophoric properties. Our results show that the sensitivity of rat heart mitochondria to Ca2+ ions was much lower in the case of MPTP opening and much higher in the case of respiration inhibition as compared to liver mitochondria.

Steric blocking upside down: a different way of thinking about the competition between myosin and tropomyosin

At low free Ca2+, the actin binding proteins tropomyosin, troponin I, troponin T and troponin C inhibit contraction in striated muscles. Ca2+ activation alters the position of tropomyosin on actin to uncover binding sites for high affinity forms of myosin (i.e., myosin-ADP). Inhibition of contraction is commonly thought to result from steric blocking of myosin binding to actin by tropomyosin. However, myosin-ADP binding to actin is energetically more favorable than localization of tropomyosin in the blocking position. Tropomyosin is an effective inhibitor of binding only at low levels of myosin-ADP. At low free Ca2+, troponin-tropomyosin also inhibits the rate of a step associated with Pi release to about 1% of the maximum rate. This results in accumulation of myosin with bound ATP and ADP-Pi. Such myosin binds weakly to actin. Ca2+ activation increases the rate of Pi release, but not to the maximum value, and increases the population of myosin-ADP. The high affinity binding of myosin-ADP to actin can displace tropomyosin into the fully active position in relation to the amount of myosin-ADP bound. It seems likely that an important outcome of the steric clash between myosin-ADP and tropomyosin is the dual activation by Ca2+ and myosin-ADP. The C-terminal region of troponin T (TnT) contributes to the incomplete activation by Ca2+ alone. Because this region of TnT is highly conserved, the ability of myosin-ADP to move tropomyosin may be more important than any restriction that tropomyosin may place on myosin binding.

Age-Dependent Changes in Calcium Regulation after Myocardial Ischemia–Reperfusion Injury

During aging, heart structure and function gradually deteriorate, which subsequently increases susceptibility to ischemia–reperfusion (IR). Maintenance of Ca2+ homeostasis is critical for cardiac contractility. We used Langendorff’s model to monitor the susceptibility of aging (6-, 15-, and 24-month-old) hearts to IR, with a specific focus on Ca2+-handling proteins. IR, but not aging itself, triggered left ventricular changes when the maximum rate of pressure development decreased in 24-month-olds, and the maximum rate of relaxation was most affected in 6-month-old hearts. Aging caused a deprivation of Ca2+-ATPase (SERCA2a), Na+/Ca2+ exchanger, mitochondrial Ca2+ uniporter, and ryanodine receptor contents. IR-induced damage to ryanodine receptor stimulates Ca2+ leakage in 6-month-old hearts and elevated phospholamban (PLN)-to-SERCA2a ratio can slow down Ca2+ reuptake seen at 2–5 μM Ca2+. Total and monomeric PLN mirrored the response of overexpressed SERCA2a after IR in 24-month-old hearts, resulting in stable Ca2+-ATPase activity. Upregulated PLN accelerated inhibition of Ca2+-ATPase activity at low free Ca2+ in 15-month-old after IR, and reduced SERCA2a content subsequently impairs the Ca2+-sequestering capacity. In conclusion, our study suggests that aging is associated with a significant decrease in the abundance and function of Ca2+-handling proteins. However, the IR-induced damage was not increased during aging.

Avoiding and allowing apatite precipitation in oxygenic photolithotrophs.

The essential elements Ca and P, taken up and used metabolically as Ca2+ and H2 PO4 - /HPO4 2- respectively, could precipitate as one or more of the insoluble forms calcium phosphate (mainly apatite) if the free ion concentrations and pH are high enough. In the cytosol, chloroplast stroma, and mitochondrial matrix, the very low free Ca2+ concentration avoids calcium phosphate precipitation, apart from occasionally in the mitochondrial matrix. The low free Ca2+ concentration in these compartments is commonly thought of in terms of the role of Ca2+ in signalling. However, it also helps avoids calcium phosphate precipitation, and this could be its earliest function in evolution. In vacuoles, cell walls, and xylem conduits, there can be relatively high concentrations of Ca2+ and inorganic orthophosphate, but pH and/or other ligands for Ca2+ , suggests that calcium phosphate precipitates are rare. However, apatite is precipitated under metabolic control in shoot trichomes, and by evaporative water loss in hydathodes, in some terrestrial flowering plants. In aquatic macrophytes that deposit CaCO3 on their cell walls or in their environment as a result of pH increase or removal of inhibitors of nucleation or crystal growth, phosphate is sometimes incorporated in the CaCO3 . Calcium phosphate precipitation also occurs in some stromatolites.

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45μmol/g of body weight) twice a day for a period of 2weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca2+, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg2+-bound CaM (MgCaM). Upon titration of Ca2+ into MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca2+-binding domains, EF3 and EF4 (CaKD = 60 ± 7 nM). As higher concentrations of free Ca2+ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (CaKD = 1000 ± 160 nM). Thermodynamic and kinetic Ca2+ binding studies showed that BP2 addition increased the Ca2+-binding affinity of CaM and slowed its Ca2+ dissociation rates (koff) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca2+ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca2+ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca2+-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca2+-loaded CaM (CaCaM-BP2). Because this structural rearrangement observed for the CaCaM-BP2 complex occurs as intracellular free Ca2+ concentrations approach those typical of a Ca2+-signaling event (CaKD = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaCaM-STRA6.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Ca2+ levels in Daphnia hemolymph may explain occurrences of daphniid species along recent Ca gradients in Canadian soft-water lakes.

Calcium levels are declining in eastern North American and western European lakes. This widespread issue is affecting the composition of crustacean zooplankton communities, as the presence and abundance of several calcium-rich daphniid species are declining, while two other daphniids, D. catawba and D. ambigua, that apparently tolerate low calcium environments, are prospering. The physiological basis for low calcium tolerance of these daphniids is unknown. In this study the presence of one Ca-rich (D. pulicaria) and one Ca-poor (D. ambigua) daphniid species in Canadian Shield lakes is assessed in relation to lake water Ca levels. The occurrence of D. ambigua was independent of Ca levels in Ontario lakes, whereas D. pulicaria was more likely to occur in lakes with relatively more Ca. In the laboratory, D. ambigua maintained lower levels of hemolymph Ca2+ across a range of low Ca levels (0.7 to 7 mg l−1) compared with D. pulicaria. The hemolymph pH remained steady across this Ca gradient in D. ambigua while it was significantly more acidic in D. pulicaria in the two lowest Ca treatments. While Ca2+ uptake was observed adjacent to the surface of D. ambigua individuals, Ca2+ loss was observed for D. pulicaria assayed under moderately high Ca levels. Based on these observations we propose that D. ambigua is able to survive in low Ca lakes by maintaining low free ionic Ca2+ levels in the hemolymph which minimizes the Ca gradient across the body wall in low Ca water thus limiting overall Ca loss and facilitating Ca2+ uptake.

The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate "C" State of Regulated Actin under High- and Low-Free Ca(2+) Conditions.

The R146G mutation of troponin I (TnI) is associated with hypertrophic cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear to be linked to changes in regulated actin distributions, we determined the extent to which the R146G TnI mutant alters the distribution of states at low and high Ca(2+) concentrations. We show, from measurements of the kcat for actin-activated ATPase activity at saturating Ca(2+) concentrations, that R146G TnI reduced the population of the active, M, state to 25% of the wild-type level. Together with acrylodan-tropomyosin fluorescence measurements of the B state, it appeared that the C state was populated at ∼91% of the total for the R146G TnI-containing actin filaments. The C state was also more heavily populated at low Ca(2+) concentrations. Acrylodan-tropomyosin fluorescence changes showed a large diminution in the inactive state value relative to the wild-type value without a comparable increase in the active state. Furthermore, the rate of binding of rigor S1 to pyrene-labeled actin filaments containing R146G TnI was faster than the rate of binding to wild-type filaments at low free Ca(2+) concentrations. These results indicate that the inhibitory region of TnI affects the B-C and M-C equilibria of actin-tropomyosin-troponin. The observation that a mutation in the inhibitory region affects the M-C equilibrium may point to a novel regulatory interaction.

Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms.

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm(-2) lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl2 or 2 mM MgCl2 significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na(+) and low free Ca(2+) and Mg(2+) concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.

Altered Ion Channel Properties of Ryanodine Receptor from Heart Mice Lacking Calstabin2

The cardiac ryanodine receptor (RyR2) is the main channel for the release of intracellular calcium during excitation-contraction coupling in cardiac muscle. Calstabin2 (i.e. FKBP12.6), a member of the FK506-binding protein family, has high affinity to RyR2, stabilizing RyR2 in the closed state and reducing its activity. The goal of the present work was first to examine the ion channel properties of RyR2 from calstabin2-/- mice reconstituted into lipid bilayers in resting condition. The second aim was to evaluate the effect of adding recombinant calstabin2 on RyR2 ion channel properties. RyR2 channels from 9 months old calstabin2 deficient mice exhibited an increase of opening probability compared to RyR2 from wild-type mice in conditions of low free cytosolic Ca2+ of 150 nM to approximate the conditions in the heart during diastole. The activity of RyR2 calstabin2-/- channels was also higher with 350 nmol/L and 700 nmol/L cytosolic Ca2+. When channels were activated by higher cytosolic Ca2+ concentration (> 700 nmol/L free Ca2+), no differences in open probability were found between RyR2 calstabin2-/- and WT channels. Addition of 1 mmol/L Na2-ATP increased the number of long openings to the maximum conductance in both native and calstabin2-/- channels with a prevalent sub-conductance activity for the depleted calstabin2 RyR2 channels. Addition of calstabin2 did neither increase nor decrease RyR2 calstabin2-/- open probability and had no effect in preventing the sub-conductance activity. This suggests that in vitro addition of calstabin2 is not sufficient to restore the RyR2/calstabin2 complex. This conclusion should further supported by the biochemical analyzes of RyR2 macromolecular complex remodeling, performed along with age-dependency.

DREAM regulates insulin promoter activity through newly identified DRE element

AbstractDownstream regulatory element antagonist modulator (DREAM) protein is a 31 kDa Ca2+-regulated transcriptional repressor. It functions as a silencer of the gene transcription. In low intracellular free Ca2+ concentration DREAM tightly binds to the downstream regulatory element (DRE) of gene promoter and impedes the transcription. In higher Ca2+ concentrations DREAM binds Ca2+ and disconnects from DRE of the gene promoter enabling transcription. We report that DREAM is expressed in different human tissues including the pancreas, where it is located in the islets of Langerhans. Location of DREAM in RIN-F5 cells in cultures is restricted to the nucleus and membranes and changes after increased Ca2+-levels. The proteins dissociate from dimmers to monomers and translocate out of the nucleus. The expression of DREAM in β-cells in the islets of Langerhans regulates the promoter activity of the insulin gene by directly interacting with the sequence located between +52 bp and +81 bp downstream of the transcriptional start site of the promoter. Our results provide evidence for the existence of DRE sequence in the insulin gene promoter. It is suggested that DREAM is a repressor of insulin gene transcription, whose effect is mediated by direct binding to DRE sequence.

Motility of astrocytic mitochondria is arrested by Ca2+-dependent interaction between mitochondria and actin filaments

Motility of mitochondria, as well as their activity-dependent immobilization (“trapping”), is essential for neuronal function, but its regulation by cytoskeleton and relevance for glial cell signalling are unknown. Using time-lapse fluorescence imaging in rat cultured astrocytes, we evaluated the role of microtubules and actin filaments in motility of mitochondria in resting cells and during physiological or pathological Ca2+ elevations. We found that mitochondria were significantly more aligned with microtubules than with actin filaments. Mitochondria were highly mobile under resting conditions at low intracellular free Ca2+ concentrations ([Ca2+]i). Activation of a moderate increase in [Ca2+]i by either low-dose ionomycin or ATP immobilized mitochondria significantly but reversibly, without affecting mitochondrial morphology. A larger dose of ionomycin caused irreversible arrest and fragmentation of mitochondria. Disruption of microtubules completely arrested mitochondrial motility, while disruption of actin filaments had no effect on the basal mitochondrial motility at resting [Ca2+]i levels but significantly reduced mitochondrial immobilization during [Ca2+]i elevations. These results suggest that: (i) motility of astrocytic mitochondria is inversely related to [Ca2+]i, (ii) mitochondria require intact microtubules for their motility, and (iii) elevated [Ca2+]i immobilizes mitochondria by strengthening their interaction with actin filaments.

Making sense out of Ca2+signals: their role in regulating stomatal movements

Plant cells maintain high Ca(2+) concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca(2+) concentrations, most likely provided the backbone for the development of Ca(2+)-dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca(2+) homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca(2+) concentration, are linked to Ca(2+)-based signalling. A large number of environmental stimuli can evoke Ca(2+) signals, but the Ca(2+)-induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca(2+)-dependent responses. A signal may evoke a specific Ca(2+) signature that is recognized by downstream signalling components. Alternatively, Ca(2+) signals are accompanied by Ca(2+)-independent signalling events that determine the specificity of the response. The existence of such parallel-acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca(2+) signals. Future research may shed new light on the relation between parallel acting Ca(2+)-dependent and -independent events, and may provide insights in their evolutionary origin.

Relaxing effects of 17(18)-EpETE on arterial and airway smooth muscles in human lung

Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an omega-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known BK(Ca) and K(ATP) channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 muM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca(2+) sensitivity of the myofilaments, since free Ca(2+) concentration-response curves, performed on beta-escin-permeabilized cultured explants, were shifted toward higher Ca(2+). Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.

FRET Conformational Analysis of Calmodulin Binding to Nitric Oxide Synthase Peptides and Enzymes

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.

Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25, Syntaxin1 and VAMP in human parathyroid tissue

In regulated exocytosis synaptosomal-associated protein of 25kDa (SNAP-25) is one of the key-players in the formation of SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex and membrane fusion. SNARE proteins are essentially expressed in neurons, neuroendocrine and endocrine cells. Whether parathyroid cells express these proteins is not known. In this study, we have examined the expression of the SNARE protein SNAP-25 and its cellular homologue SNAP-23, as well as syntaxin1 and VAMP (vesicle-associated membrane protein) in samples of normal parathyroid tissue, chief cell adenoma, and parathyroid carcinoma, using immunohistochemistry and Western blot analysis. SNAP-23 and VAMP were evenly expressed in all studied parathyroid tissues using immunohistochemistry and/or Western blot analysis. SNAP-25 (and Syntaxin1) was not expressed in normal parathyroid tissue, but in approximately 20% of chief cell adenomas, and in approximately 45% of parathyroid carcinoma samples. It is likely that the SNARE proteins SNAP-23 and VAMP play a role in the stimulus-secretion coupling and exocytosis of parathyroid hormone as these proteins were expressed in all of the parathyroid samples we studied. In particular, preferential expression of SNAP-23 rather than SNAP-25 provides an explanation of the high level of PTH secretion that occurs under conditions of low cytoplasmic free Ca(2+) concentration (around 0.1micromol/l). SNAP-25 (and Syntaxin1) appears to be a tumour-specific protein(s) in parathyroid tissues since its expression was restricted to pathological tissues.

Phosphorylation of the autoinhibitory insert in endothelial nitric oxide synthase promotes calmodulin binding at low free Ca2+ concentrations

The 45‐residue autoinhibitory insert in the endothelial nitric oxide synthase (eNOS) reductase domain inhibits electron transfer, and this effect is counteracted when CaM is bound to the enzyme. We have investigated the effects of phosphorylation at Ser‐617 and Ser‐635 in this region on the Ca2+ sensitivities of CaM binding and CaM‐dependent enzyme activation and on reductase and synthase activities. The mean EC50(Ca2+) values (± SD) for CaM binding to bovine eNOS and mutants with S617D, S635D, S617D/S635D substitutions or lacking the entire autoinhibitory insert were determined to be 220 ± 25, 109 ± 7, 202 ± 21, 117 ± 14, or 47 ± 9 nM, respectively. Corresponding mean EC50(Ca2+) values for eNOS activation were found to be 440 ± 55, 240 ± 28, 461 ± 56, 263 ± 28, or 124 ± 16 nM. The S617D substitution and deletion of the insert were found to increase CaM‐dependent synthase and cytochrome c reductase activities 2‐fold, and to respectively increase CaM‐independent reductase activity 3‐ and 6‐fold. The S635D substitution was found to have no significant effect on any property examined. These results indicate that phosphorylation at Ser‐617 acts synergistically with CaM to counteract the effects of the autoinhibitory insert. Given a resting free Ca2+ concentration in endothelial cells of 50 – 150 nM, phosphorylation at this site also appears to promote binding of CaM to the synthase under basal conditions.Source of research support: NIH Grant GM074887.

Expression level and activity profile of membrane bound guanylate cyclase type 2 in rod outer segments

Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca(2+)-binding proteins named GC-activating proteins that operate as Ca2+ sensors and enable cyclases to respond to changes of intracellular Ca2+after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca2+ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca2+ sensitivity of the Ca2+ sensor is controlled by the mode of target-sensor interaction.

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BK(Ca) channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 microM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentrations. Together, these results indicate that 20-HETE-dependent activation of BK(Ca) channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.