Decreased Calcium Uptake Research Articles

Calcium burns beige.

In this issue of Journal of Experimental Medicine, Yin et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20212491) discover that loss of FNIP1 is associated with browning of white adipose tissue, which they propose is driven by decreased calcium uptake into the ER.

Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant.

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6. We show that both the G660R and R510stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.

Programmed Cell Death 5 Provides Negative Feedback on Cardiac Hypertrophy Through the Stabilization of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a Protein.

PDCD5 (programmed cell death 5) is ubiquitously expressed in tissues, including the heart; however, the mechanism underlying the cardiac function of PDCD5 has not been understood. We investigated the mechanisms of PDCD5 in the pathogenesis of cardiac hypertrophy. Cardiac-specific PDCD5 knockout mice developed severe cardiac hypertrophy and impaired cardiac function, whereas PDCD5 protein was significantly increased in transverse aortic constriction mouse hearts and phenylephrine-stimulated cardiomyocytes. Overexpression of PDCD5 inhibited phenylephrine-induced cardiomyocyte hypertrophy, and knockdown of PDCD5 induced cardiomyocyte hypertrophy and aggravated phenylephrine-induced hypertrophy. The expression of PDCD5 protein was regulated by NFATc2 (nuclear factor of activated T cells c2) during hypertrophy. SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression was decreased in PDCD5-deficient mouse hearts because of increased ubiquitination. PDCD5-deficient cardiomyocytes displayed decreased calcium uptake rate, slowed decay of Ca2+ transients, decreased calcium stores, and diastolic dysfunction. Moreover, reintroduction of PDCD5 in PDCD5-deficient mouse hearts reserved SERCA2a protein, suppressed NFATc2 protein, and rescued the hypertrophy and cardiac dysfunction. Our results revealed that PDCD5 is a novel target of NFATc2 in the hypertrophic heart and provides negative feedback to protect the heart against excessive hypertrophy via the stabilization of SERCA2a protein.

Ocean acidification: effects of pH on 45Ca uptake by lobster branchiostegites.

Gill chambers of the Atlantic lobster, Homarus americanus, possess three structures that are involved with respiration and ion regulation: gill filaments, epipodites, and branchiostegites. This paper describes ion transport mechanisms present in the plasma membranes of branchiostegite epithelial cells and the effects of pH on the uptake of 45Ca by these processes. Partially purified membrane vesicles (PPMV) of branchiostegite cells were produced by a homogenization/centrifugation method that has previously been used to define ion transport processes in both crab and lobster gill tissues. In the present study, lobster branchiostegite PPMV 45Ca uptake was highest at pH 8.5 and lowest at pH values between 6.0 and 7.0 (p < 0.02). At pH 8.0, 45Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, 45Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K+-induced membrane potential (PD, inside negative) doubled 45Ca uptake at pH 7.0 above that in the absence of a PD (p < 0.05). An induced PD at pH 8.0 did not significantly (p > 0.05) affect 45Ca uptake observed in the absence of a PD, but was threefold greater than uptake at pH 7.0 in the absence of a PD (p < 0.05). Amiloride (2mM) did not affect 45Ca uptake at pH 8.0, but 2mM amiloride + 100µM verapamil reduced uptake by approximately 50%. In the presence of both 2mM amiloride + 100µM verapamil, 15s 45Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5mM) (Km = 4.2 ± 0.3mM; Jmax = 9792 ± 439pmol/mg protein × 15s). 45Ca influxes at pH 7.5 under the same conditions were also hyperbolic with Km = 8.3 ± 1.4mM; Jmax = 10732 ± 1250pmol/mg protein × 15s. Km values were significantly different (p < 0.05), but Jmax values were not (p > 0.05). These results suggest that 45Ca uptake by lobster branchiostegites may have occurred by the combination of diffusion through a verapamil-inhibited calcium channel and carrier-mediated transport by amiloride-insensitive, electroneutral, 1Ca2+/2H+ antiporters. Decreased pH, as might occur during ocean acidification, did not appear to modify calcium diffusion through the channels, but protons acted as competitive inhibitors of calcium transport by carrier-mediated antiport. Decreased calcium uptake with continued ocean acidification may significantly affect calcification processes during periodic molting, potentially influencing mortality.

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids.

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.

Zinc Improves the Bone Mechanical Strength in Ovariectomized Rat Model by Restoring Bone Composition and Hydroxyapatite Crystallite Dimension

Purpose: Osteoporosis is a bone metabolic disorder which is well known to increase bone porosity and is the outcome of various factors like ageing, genetic, nutritional deficiency, decreased calcium uptake, and last but the most important hormonal imbalances. Hormonal imbalance is one of the major factors affecting women worldwide and leading to osteoporosis. Trace elements play a very essential role in number of pathological conditions. Ingestion of zinc in the early stages of bone loss may be more beneficial in mitigating bone loss and also in improving the overall strength of the bone. In the current work, we have intended to extract the information pertaining to the mechanical strength of bone, bone tissue composition and hydroxyapatite crystallite size upon supplementing zinc in the osteopenic condition. Methods: Forty eight wistar female rats in two set of twenty four animals each were assigned to four groups: Control, Zinc, Ovariectomized (OVX) and OVX+Zinc. Duration of the treatment period was of eight weeks. Biochemical estimations were carried out to make comparison between the treatment groups based on bone metabolism markers in serum. Bone mechanical strength of both the bones i.e., femur and tibia, was assessed using texture analyzer. Also, bone matrix analysis using Fourier transformer infrared spectroscopy and X-ray diffraction studies were carried out for all the treatment groups. Results: Estradiol levels decreased and tartarate-resistant acid phosphatase 5b levels increased in the OVX group. Zinc supplemented following ovariectomy regulated these levels. The OVX group showed significantly higher serum alkaline phosphatase levels, which recovered upon zinc supplementation. Further, zinc plays a potential role in preventing bone tissue deterioration by restoring its composition and microstructure in the post-menopausal condition, thereby, maintaining the mechanical strength of the bone. Conclusion: These findings suggested that alterations in the bone tissue material properties following estrogen deficiency can be averted by zinc if administered at early stages of bone loss.

Bone mineral density and expression of vitamin D receptor-dependent calcium uptake mechanisms in the proximal small intestine after bariatric surgery.

Roux-en-Y gastric bypass may lead to impaired calcium uptake. Therefore, operation-specific effects of gastric bypass and vertical banded gastroplasty on bone mineral density (BMD) were examined in a randomized clinical trial. Bone resorption markers and mechanisms of decreased calcium uptake after gastric bypass were investigated using blood and endoscopic samples from two additional patient cohorts. Total BMD and non-weight-bearing skull BMD were measured by dual-energy X-ray absorptiometry at baseline, and 1 and 6 years after gastric bypass or vertical banded gastroplasty in patients who were not receiving calcium supplements. Bone resorption markers in serum and calcium uptake mechanisms in jejunal mucosa biopsies were analysed after gastric bypass by proteomics including radioimmunoassay, gel electrophoresis and mass spectrometry. One year after surgery, weight loss was similar after gastric bypass and vertical banded gastroplasty. There was a moderate decrease in skull BMD after gastric bypass, but not after vertical banded gastroplasty (P < 0·001). Between 1 and 6 years after gastric bypass, skull BMD and total BMD continued to decrease (P = 0·001). C-terminal telopeptide levels in serum had increased twofold by 18 months after gastric bypass. Proteomic analysis of the jejunal mucosa revealed decreased levels of heat-shock protein 90β, a co-activator of the vitamin D receptor, after gastric bypass. Despite increased vitamin D receptor levels, expression of the vitamin D receptor-regulated calcium transporter protein TRPV6 decreased. BMD decreases independently of weight after gastric bypass. Bone loss might be attributed to impaired calcium absorption caused by decreased activation of vitamin D-dependent calcium absorption mechanisms mediated by heat-shock protein 90β and TRPV6.

Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 μM kainic acid (KA) for 1–24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced 45Ca2+ uptake. The maximum effect (70–80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and 45Ca2+ uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4.

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.

Anti-allergic activity of grapeseed extract (GSE) on RBL-2H3 mast cells

Grapeseed extract (GSE) is a rich source of natural phenolic compounds and possesses various pharmacological activities, including antioxidant, anticarcinogenic and anti-inflammatory properties. However, effects of GSE on immunoglobulin (Ig) E-mediated allergic responses still remain elusive. In the present study, the effects of GSE on activation and degranulation of RBL-2H3 mast cells were investigated. GSE pretreatments (20–100μg/ml) reduced IgE-antigen mediated release of β-hexosaminidase and histamine in RBL-2H3 cells. Additionally, GSE reversibly inhibited expression of FcεRI on RBL-2H3 cells. GSE treatments caused a significant elevation of intracellular cAMP levels, whereas the Ca2+ influx upon antigen stimulation was inhibited. Suppression on FcεRI expression together with decreased calcium uptake and increased cAMP level might be involved in attenuated degranulation of mast cells by GSE treatment. Our results suggest a possible pharmaceutical application of GSE in treating type I allergic diseases.

EFFECTS OF ROOT ZONE PH AND NUTRIENT CONCENTRATION ON THE GROWTH AND NUTRIENT UPTAKE OF TOMATO SEEDLINGS

The effects of nutrient concentration and pH, two major chemical properties of soil, on plant responses were investigated with seedlings of tomato, which is widely grown in greenhouses, as the model plant. An experiment with four levels of nutrient concentration [None (NC 0), 1 (NC 1), 5 (NC 5), and 10 folds (NC 10)], in combination with three pH levels (4, 6, and 8), was conducted. The fresh and dry weight and leaf area of tomato seedling increased until NC5, but decreased at NC10. Transpiration rates, stomatal conductance, and evapotranspiration reduced with increasing nutrient concentration. The responses to the pH levels differed by the nutrient-concentration levels. The unsuitable root zone pH, pH 4 and pH 8, caused more reductions of transpiration rates, stomatal conductance, and evapotranspiration than pH 6 does under high nutrient concentrations, specifically pH 8 strongly depressed these. At pH 8, fresh and dry weight and areas of shoot and root sharply reduced more than at pH 4. Total nitrogen (N) content in shoot and root was not significantly affected by pHs and nutrient concentrations. High pH and high nutrient concentration level increased magnesium uptake, but decreased calcium uptake. The contents of microelements decreased as nutrient concentration increased. Changes of macro and micro element content among treatments were smaller in shoot than in root. The results imply that not only nutrient concentration but also root zone pH is associated with osmotic stress, and tomato seedlings have a tendency to reduce shoot more than root against the stress. However, nutrient contents of shoot are maintained over a certain level and surplus nutrients are stored at the root.

Effect of Chlorogenic acid on mast cell-dependent anaphylactic reaction

Chlorogenic acid (CGA), a naturally occurring polyphenol compound, has a number of biological activities. However, roles of CGA in the mast cell-dependent anaphylactic reaction have not been fully examined. In the present study, the effect and mechanism of CGA on mast cell-dependent anaphylactic reaction were investigated using in vivo and in vitro models. CGA inhibited compound 48/80-induced systemic anaphylactic shock in mice and skin vascular permeability in rats. CGA also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis (PCA). Moreover, CGA dose-dependently reduced histamine and TNF-α release from RBL-2H3 cells activated by anti-DNP IgE. Pretreatment with CGA suppressed IgE–antigen complex induced calcium uptake into RBL-2H3 cells. When CGA was added, the level of intracellular cyclic adenosine monophosphate (cAMP) in RBL-2H3 cells was significantly elevated compared with the untreated cells. Decreased calcium uptake and increased cAMP level might be involved in the inhibitory effect of CGA on mast cell activation. These results suggest a possible therapeutic application of CGA in allergic diseases.

Silibinin Attenuates Mast Cell-Mediated Anaphylaxis-Like Reactions

Silibinin is known to have hepatoprotective, anti-carcinogenic and anti-inflammatory effects. However, roles of silibinin in the immediate-type allergic reactions (anaphylaxis) have not fully been investigated. In the present study, we have demonstrated that silibinin attenuated mast cell-mediated anaphylaxis-like reactions involved in allergic diseases. Oral administration of silibinin inhibited compound 48/80-induced passive cutaneous anaphylaxis-like reaction in mice. Silibinin also attenuated anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-mediated passive systemic and cutaneous anaphylaxis. Silibinin had no cytotoxicity on rat peritoneal mast cells (RPMC). Silibinin dose-dependently reduced histamine release from RPMC activated by compound 48/80 or anti-DNP IgE. Moreover, silibinin inhibited the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 in RPMC. Pretreatment of silibinin suppressed the antigen-stimulated calcium uptake and activation of nuclear factor-kappa B (NF-kappaB) in RPMC. Furthermore, silibinin increased the intracellular cAMP level. Increased cAMP, decreased calcium uptake and suppressed NF-kappaB activity might be involved in the inhibitory effect of silibinin on the secretory response. Our findings provide possibility that silibinin may serve as an effective therapeutic agent for allergic diseases.

Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel

The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.

Early mitochondrial alterations in ATRA-induced cell death

All-trans retinoic acid (ATRA) induces differentiation and subsequent apoptosis in a variety of cell lines. Using the myeloid cell line P39, we show that ATRA disturbs mitochondrial functional activity long before any detectable signs of apoptosis occur. These early changes include diminished mitochondrial oxygen consumption, decreased calcium uptake by mitochondria and as a result, a lower mitochondrial matrix calcium concentration. Granulocyte colony-stimulating factor (G-CSF) increases mitochondrial respiration and calcium accumulation capacity and subsequently blocks ATRA-induced apoptosis. Nifedipine, a plasma membrane calcium channel blocker, inhibits apoptosis-related changes, such as the loss of the mitochondrial membrane potential and activation of caspases. Thus, the properties of ATRA and G-CSF to modulate mitochondrial respiration and intracellular calcium control are novel findings, which give insight into their precise molecular mode of action.

A Family of Basic Amino Acid Transporters of the Vacuolar Membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

Clinical characteristic of adynamic bone

Suppression on both bone formation and absorption is a characteristic histology of adynamic (or aplastic) bone (ABD). This new entity of renal osteodystrophy started to appear in bone biopsy findings in 1983. Most significant etiology of adynamic bone is relatively lower serum PTH level. Any study has failed to demonstrate that ABD would increase the incidence of bone fracture in dialysis patients. However, ABD increases the risk of hypercalcemia because a decrease in calcium uptake by bone and might shorten the life span of dialysis patients.

Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ.

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.

Global ischemia-induced inhibition of the coupling ratio of calcium uptake and ATP hydrolysis by rat whole brain microsomal Mg 2+/Ca 2+ ATPase

Ischemia is associated with a loss of cytosolic calcium homeostasis. Intracellular stores, particularly in endoplasmic reticulum, are critical for the maintenance of calcium homeostasis. Recent studies have shown that ischemia significantly inhibited microsomal calcium uptake mediated by Mg 2+/Ca 2+ ATPase, the major mechanism of endoplasmic reticulum calcium sequestration. This study was initiated to determine whether the decreased calcium uptake caused by ischemia was the result of inhibition of Mg 2+/Ca 2+ ATPase activity or an uncoupling of calcium uptake from ATP hydrolysis. The microsomal Mg 2+/Ca 2+ ATPase specific inhibitor thapsigargin partially inhibited ATPase activity and completely inhibited calcium uptake. ATPase inhibited by thapsigargin was considered microsomal Mg 2+/Ca 2+ ATPase. Ischemia from 5 to 60 min had no significant effect on thapsigargin sensitive ATPase activity. However, under identical conditions, increasing ischemia from 5 to 60 min significantly inhibited microsomal calcium uptake. Comparing calcium uptake to ATP hydrolysis as ischemia increased from 5 to 60 min revealed that the coupling ratio of calcium molecules sequestered to ATP molecules hydrolyzed became significantly decreased. The results demonstrated that the effect of ischemia on microsomal calcium uptake was mediated by an uncoupling of calcium transport from Mg 2+/Ca 2+ ATPase activity.

Sarcoplasmic reticulum responses to repeated sprints are affected by conditioning of horses.

Sarcoplasmic reticulum (SR) responses to repeated sprints and to physical conditioning were studied in 10 Quarter Horses. Exercise tests (four repeated sprints on a treadmill) were conducted before and after 12 wk of sprint conditioning. Muscle samples from the middle gluteal muscle were taken before and after each exercise test, and SR vesicles were isolated. Calcium uptake was determined spectrophotometrically using antipyrylazo III, and Ca2+-ATPase activity was determined using an enzyme-linked optical assay. Conditioning increased calcium uptake rate and Ca2+-ATPase activity by 14 and 38%, respectively, before exercise and by 25 and 26% after exercise. Exercise decreased calcium uptake rate and Ca2+-ATPase activity by 37 and 27%, respectively, before conditioning and by 28 and 21% after conditioning. Decreases in calcium uptake and Ca2+-ATPase activity of SR have been associated with fatigue during exercise, and this association is strengthened by the moderating effect of conditioning.