Diaphragm single-fiber weakness and loss of myosin in congestive heart failure rats

Diaphragm weakness commonly occurs in patients with congestive heart failure (CHF) and is an independent predictor of mortality. However, the pathophysiology of diaphragm weakness is poorly understood. We hypothesized that CHF induces diaphragm weakness at the single-fiber level by decreasing myosin content. In addition, we hypothesized that myofibrillar Ca(2+) sensitivity is decreased and cross-bridge kinetics are slower in CHF diaphragm fibers. Finally, we hypothesized that loss of myosin in CHF diaphragm weakness is associated with increased proteolytic activities of caspase-3 and the proteasome. In skinned diaphragm single fibers of rats with CHF, induced by left coronary artery ligation, maximum force generation was reduced by approximately 35% (P < 0.01) compared with sham-operated animals for slow, 2a, and 2x fibers. In these CHF diaphragm fibers, myosin heavy chain content per half-sarcomere was concomitantly decreased (P < 0.01). Ca(2+) sensitivity of force generation and the rate constant of tension redevelopment were significantly reduced in CHF diaphragm fibers compared with sham-operated animals for all fiber types. The cleavage activity of the proteolytic enzyme caspase-3 and the proteasome were approximately 30% (P < 0.05) and approximately 60% (P < 0.05) higher, respectively, in diaphragm homogenates from CHF rats than from sham-operated rats. The present study demonstrates diaphragm weakness at the single-fiber level in a myocardial infarct model of CHF. The reduced maximal force generation can be explained by a loss of myosin content in all fiber types and is associated with activation of caspase-3 and the proteasome. Furthermore, CHF decreases myofibrillar Ca(2+) sensitivity and slows cross-bridge cycling kinetics in diaphragm fibers.     

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Differential effects of aluminum ion on smooth muscle calpain I and calpain II activities.

1. In millimolar Ca2+, smooth muscle calpains I and II were inhibited by aluminum ion. 2. At sub-millimolar Ca2+, calpain II, but not calpain I, was activated by low millimolar aluminum ion. 3. Calpastatin inhibited aluminum ion-activated calpain II. 4. Aluminum ion-activated and Ca(2+)-activated calpain II gave almost identical patterns of desmin cleavage. 5. Aluminum-activated calpain II, unlike the Ca(2+)-activated enzyme, did not autolyze and retained its proteolytic activity over extended periods of time.     

Effect of mu-calpain on m-calpain

The free Ca(2+) concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 microM and are much higher than the 50-500 nM free Ca(2+) concentrations that exist in living cells. Consequently, a number of studies have attempted to find mechanisms that would lower the Ca(2+) concentration required for proteolytic activity of m-calpain. Although autolysis lowers the Ca(2+) concentration required for proteolytic activity of m-calpain, 90-400 microM Ca(2+) is required for a half-maximal rate of autolysis of m-calpain, even in the presence of phospholipid. It has been suggested that mu-calpain, which has a lower Ca(2+) requirement than m-calpain, might proteolyze m-calpain and reduce its Ca(2+) requirement to a level that would allow it to be active at physiological Ca(2+) concentrations. We have incubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain:m-calpain, in the presence of 50 microM free Ca(2+); this Ca(2+) concentration is high enough for more than half-maximal activity of mu-calpain, but does not activate m-calpain. Under these conditions, mu-calpain caused no detectable proteolytic degradation of the m-calpain polypeptide and did not change the Ca(2+) concentration required for proteolytic activity of m-calpain. mu-Calpain also did not degrade the m-calpain polypeptide at 1000 microM Ca(2+), which is a Ca(2+) concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1000 microM Ca(2+) and because the subsite specificities of mu- and m-calpain are very similar if not identical, failure of mu-calpain to rapidly degrade m-calpain at 1000 microM Ca(2+) suggests a unique role of autolysis in calpain function.     

Calcium fluxes in human trophoblast (BeWo) cells: calcium channels,calcium-ATPase,and sodium-calcium exchanger expression

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are poorly understood. In the current study, we have investigated the characteristics of Ca(2+) fluxes in relation with cell Ca(2+) homeostasis in the human placental trophoblast cell line BeWo. Time-courses of Ca(2+) uptake by BeWo cells displayed rapid initial entry (initial velocity (V(i)) of 3.42 +/- 0.35 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid declined of cell-associated (45)Ca(2+) with a V(i) of efflux (Ve(i)) of 3.30 +/- 0.08 nmol/mg protein/min. Further identification of membrane gates for Ca(2+) entry in BeWo cells was carried out. Expression of Ca(2+) transporter/channel CaT1 and L-type alpha(1S) subunit was showed by RT-PCR. However, mRNA for CaT2 channel and L-type alpha(1C) and alpha(1D) subunits were not revealed. Membrane systems responsible for intracellular Ca(2+) extrusion from BeWo cells were also investigated. Plasma membrane Ca(2+)-ATPases (PMCA) and Na/Ca exchangers (NCX) were detected by Western blot in BeWo cells. Expression of specific isoforms of PMCA and NCX was further investigated by RT-PCR. Messenger RNAs of four isoforms of PMCA (PMCA 1-4) were detected. The presence of messenger RNAs of two NCX isoforms (NCX1 and NCX3) was observed. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. Inorganic ions such as cadmium and manganese did not modify the Ca(2+) fluxes, however, barium increased cell-associated (45)Ca(2+) by, in part, by reducing radiolabel exit.     

Involvement of the plasma membrane Ca2+-ATPase in the short-term response of Arabidopsis thaliana cultured cells to oligogalacturonides

Treatment of Arabidopsis thaliana cells with oligogalacturonides (OG) initiates a transient production of reactive oxygen species (ROS), the concentration of which in the medium peaks after about 20 min of treatment. The analysis of OG effects on Ca (2+) fluxes shows that OG influence both Ca (2+) influx and Ca (2+) efflux (measured as (45)Ca (2+) fluxes) in a complex way. During the first 10 - 15 min, OG stimulate Ca (2+) influx and decrease its efflux, while at successive times of treatment, OG cause an increase of Ca (2+) efflux and a slight decrease of its influx. Treatment with sub- micro M concentrations of eosin yellow (EY), which selectively inhibits the Ca (2+)-ATPase of plasma membrane (PM), completely prevents the OG-induced increase in Ca (2+) efflux. EY also suppresses the transient feature of OG-induced ROS accumulation, keeping the level of ROS in the medium high. The biochemical analysis of PM purified from OG-treated cells indicates that treatment with OG for 15 to 45 min induces a significant decrease in Ca (2+)-ATPase activation by exogenous calmodulin (CaM), and markedly increases the amount of CaM associated with the PM. During the same time span, OG do not influence the expression of At-ACA8, the main isoform of PM Ca (2+)-ATPase in suspension-cultured A. thaliana cells, and of CaM genes. Overall, the reported results demonstrate that the PM Ca (2+)-ATPase is involved in the response of plant cells to OG and is essential in regulation of the oxidative burst.     

Naproxen affects Ca(2+) fluxes in mitochondria, microsomes and plasma membrane vesicles

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca(2+) ions, including the metabolic responses of the liver to Ca(2+)-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca(2+) fluxes. The effects of naproxen on 45Ca(2+) fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45Ca(2+) and inhibited also ATP-dependent 45Ca(2+) uptake by microsomes. Naproxen did not modify 45Ca(2+) uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca(2+) efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca(2+) ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca(2+) ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca(2+)-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca(2+) through a direct action on the plasma membrane Ca(2+) influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.     

Effects of moderate heart failure and functional overload on rat plantaris muscle.

It is thought that changes in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) of skeletal muscle contribute to alterations in skeletal muscle function during congestive heart failure (CHF). It is well established that exercise training can improve muscle function. However, it is unclear whether similar adaptations will result from exercise training in a CHF patient. Therefore, the purpose of this study was to determine whether skeletal muscle during moderate CHF adapts to increased activity, utilizing the functional overload (FO) model. Significant increases in plantaris mass of the CHF-FO and sham-FO groups compared with the CHF and control (sham) groups were observed. Ca(2+) uptake rates were significantly elevated in the CHF group compared with all other groups. No differences were detected in Ca(2+) uptake rates between the CHF-FO, sham, and sham-FO groups. Increases in Ca(2+) uptake rates in moderate-CHF rats were not due to changes in SERCA isoform proportions; however, FO may have attenuated the CHF-induced increases through alterations in SERCA isoform expression. Therefore, changes in skeletal muscle Ca(2+) handling during moderate CHF may be due to alterations in regulatory mechanisms, which exercise may override, by possibly altering SERCA isoform expression.     

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.     

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.     

Calpain protects the heart from hemodynamic stress

Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.     

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

Defective Ca2+-pumping ATPase of heart sarcolemma from cardiomyopathic hamster

The Syrian cardiomyopathic hamster has a hereditary disease characterized by a progressive myocyte necrosis and intracellular calcium overload. Several systems in the heart sarcolemma that regulate the rate of Ca2+ entry or efflux were examined. There is a selective decrease of Ca2+-pumping ATPase activity in the heart sarcolemma of 40-day-old myopathic hamsters, while the Na+-Ca2+ exchange system and the ouabain-sensitive (Na+ + K+)-ATPase activity remain intact. This age-dependent decrease in Ca2+-ATPase activity closely parallels the time course of lesion development. Both the affinity for Ca2+ (Km) and the maximal velocity (Vmax) of the Ca2+-dependent ATP hydrolysis are altered. In addition, there is also an increased number of calcium channel receptor binding sites. Thus the data suggest that the imbalance in Ca2+ fluxes across the cardiac plasma membrane may be involved in the pathogenesis of this cardiomyopathy.     

Effects of methotrexate on calcium flux in rat liver mitochondria, microsomes and plasma membrane vesicles

The metabolic effects of methotrexate in perfused livers are similar to those exerted by hormones acting through Ca(2+)-dependent mechanisms. The aim of the present study was to determine whether the effects of methotrexate are mediated by a direct action on cellular Ca(2+) fluxes. Methotrexate did not affect the ATP-dependent (45)Ca(2+) uptake by mitochondria, microsomes and inside-out plasma membrane vesicles and Ca(2+) efflux from plasma membrane vesicles. However, methotrexate was able to stimulate (45)Ca(2+) release from preloaded microsomes. The amount of Ca(2+) released by methotrexate was similar to that induced by IP(3). Methotrexate could be acting through the capacitative calcium entry mechanism.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Calpain Inhibition Protects Spinal Motoneurons from the Excitotoxic Effects of AMPA In vivo

Microdialysis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in rat lumbar spinal cord produces severe motoneuron damage and consequently hindlimb paralysis. Here we studied the time course of the AMPA-induced neurodegenerative changes and motor alterations, and the protective effect of leupeptin, an inhibitor of calpain, a Ca(2+)-activated protease. Paralysis occurs at 4-6 h after AMPA perfusion, but cresyl violet staining showed that motoneuron damage starts at about 3 h and progresses until reaching 50% neuronal loss at 6 h and 90% loss at 12 h. In contrast, choline acetyltransferase (ChAT) immunohistochemistry revealed that the enzyme is already decreased at 30 min after AMPA perfusion and practically disappears at 3 h. Microdialysis coperfusion of leupeptin with AMPA prevented the motor alterations and paralysis and remarkably reduced both the decrement in ChAT immunoreactivity and the loss of motoneurons. We conclude that an increased Ca(2+) influx through Ca(2+)-permeable AMPA receptors activates calpain, and as a consequence ChAT content decreases earlier than other Ca(2+)-dependent processes, including the proteolytic activity of calpain, cause the death of motoneurons.     

Compartmental analysis of 45Ca2+ efflux in perfused rat liver: effects of hormonal stimulation.

The kinetics of calcium movements in the isolated perfused rat liver were examined using compartmental analysis of the efflux profiles of 45Ca2+ from 45Ca(2+)-equilibrated livers under a variety of calcium concentrations and hormonal treatments. From the 45Ca2+ efflux profiles, we determined that a three compartment model was appropriate to describe the movements of calcium in the liver on the time scale of the experiments. Hormonal treatment with the alpha-adrenergic agonist, phenylephrine, or the vasoactive peptide, vasopressin, during the efflux period lowered significantly the rate of transfer of Ca2+ between the internal compartments at all of the calcium concentrations employed. Also, phenylephrine treatment leads to increased transfer of Ca2+ into the liver from the perfusate. The temporal characteristics of the phenylephrine and vasopressin sensitive Ca2+ pools were examined by pulsing livers, loaded for variable periods of time with 45Ca2+, with the two hormones during the efflux of 45Ca2+ to measure the kinetics of Ca2+ exchange in the hormone-sensitive pools. Results from these experiments indicate that the rate of unstimulated Ca2+ efflux, k2, for the phenylephrine and vasopressin sensitive Ca2+ pools, modeled as a one compartment system, are the same, 0.074 and 0.078 min-1 for phenylephrine and vasopressin respectively, corresponding to half times for turnover of the pool(s) of 9.3 and 8.9 min, respectively.     

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.     

Proteolytic activity in intact sheets of polarized epithelial cells as determined by a cell-permeable fluorogenic substrate

The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell-permeable, fluorogenic calpain and proteasome substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60-70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca(2+)-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca(2+)mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.