Serum ionized magnesium and calcium levels in adult patients with seizures

OBJECTIVE: Prior studies have been equivocal about whether or not serum levels of the divalent ions calcium and magnesium are altered during different types of seizures. Magnesium is a potential modulator of seizure activity because of its ability to antagonize the excitatory calcium influx through the N-methyl-D-aspartate (NMDA) receptor. We hypothesize that serum ionized levels of calcium (Ca(2+)) and magnesium (Mg(2+)) would be altered significantly during certain types of seizures. MATERIAL AND METHODS: A convenience sample of seizure patients presenting to an emergency department (ED) were enrolled in this prospective study. Novel ion-selective electrodes were used to measure Ca(2+) and Mg(2+). Data were reported as mean values+/-standard deviations. Group comparisons were analyzed by ANOVA with post-hoc testing using the Bonferroni, or the Fisher exact test, where appropriate, alpha = 0.05 (two-tailed). RESULTS: Forty-nine patients with seizure and 32 healthy racially matched controls were included in the study. Seizure patients had a significantly (p<0.001) lower mean Mg(2+), but not total serum Mg and a significantly (p<0.001) higher Ca(2+)/Mg(2+) ratio than that in controls. CONCLUSIONS: We were able to show significantly lower Mg(2+) and higher ionized Ca(2+)/Mg(2+) ratios in seizure patients compared with a racially matched control group.     

Ionized magnesium levels and the ratio of ionized calcium to magnesium in asthma patients before and after treatment with magnesium

Objective. Prior studies have been equivocal about the efficacy of magnesium therapy in acute asthma exacerbations. We hypothesize that pretreatment ionized magnesium (Mg²⁺) levels and/or the ratio of ionized calcium to ionized magnesium (Ca²⁺/Mg²⁺) may have been confounding variables in these previous studies. Here, we report on the incidence of abnormal divalent ion levels in our asthma population. Material and methods. The study was designed as a randomized, double‐blind, placebo‐controlled trial of intravenous magnesium. Inclusion criteria were: age >18 years, percentage predicted forced expiratory volume (FEV₁) <75 % after an initial beta‐agonist. African–American patients (AA) at an urban university hospital were randomized to 2?g IV Mg or placebo. Mg²⁺ and Ca²⁺/Mg²⁺ levels were measured pre‐ and post‐infusion. Data were reported as means±SD. Student's t‐test and Fisher's exact test were used where appropriate (α = 0.05, two tailed) Results. Fifty‐five AA patients (mean age of 42.7 years±15.6 years, range18–75 years) were studied. A significantly (p<0.05) lower level of Mg²⁺ was found in asthma (AS) patients compared with that in the AA group, by 0.03?mmol/L (95 % CI, 0.007–0.053?mmol/L). The AS group had a mean increase in Ca²⁺/Mg²⁺ ratios over the AA group, of 0.27 (95 % CI, 0.16–0.38); 100 % of patients with abnormal divalent ion levels were corrected with IV magnesium. Conclusions. We identified a subgroup of asthmatic patients with significant abnormalities in their divalent ion concentrations, which was corrected with IV magnesium.     

Total and ionized serum magnesium in critically ill patients

Objective To assess the alterations in total serum magnesium (tsMg) and ionized serum magnesium (Mg²⁺) and their association with prognosis in critically ill patients.Design and setting Prospective, cohort study in the intensive care unit (ICU) of a university teaching hospital.Patients Adult patients admitted to the ICU without previous factors influencing magnesium homeostasis were included during a 6-month period.Measurements and results One hundred forty four patients were included. Mean age was 60.6±15.4 years; mean APACHE II score was 12.6±6.9. Blood samples were collected in the first 24 h after ICU admission and again on the second, third, and last days of stay in the ICU. At ICU admission 52.5% had total hypomagnesemia and 13.5% total hypermagnesemia; with respect to the Mg²⁺ 9.7% showed ionized hypomagnesemia and 23.6% ionized hypermagnesemia. Patients who developed ionized hypermagnesemia had higher mortality than patients without ionized hypermagnesemia development (P=0.04). A moderate correlation between tsMg and Mg²⁺ concentrations was found; however, a number of patients with total hypomagnesemia (69–85% during the study) had ionized normomagnesemia. The measure of agreement between tsMg and Mg²⁺ levels was poor.Conclusions Magnesium alterations are frequently found in critically ill patients. The usually determined tsMg levels are not a reflection of Mg²⁺ levels. Development of ionized hypermagnesemia is associated with prognosis.     

Evaluation of AVL988/4 analyzer for measurement of ionized magnesium and ionized calcium

Ionized magnesium (Mg⁺⁺) and ionized calcium (Ca⁺⁺) are the physiologically active forms of these elements in the body and their concentrations have clinical value. Though the AVL988/4 instrument that measures concentrations of Mg⁺⁺ and Ca⁺⁺ has been evaluated, some clinically important parameters were unknown. In this study, we evaluated AVL988/4 analyzer for measuring Mg⁺⁺ and Ca⁺⁺ concentrations and provided the following information: (1) The newly formulated Becton Dickinson (BD) Vacutainer plastic tubes with clot activator and silicone as the stopper lubricant (serial no. 367820) caused a significant high bias for the Mg⁺⁺ measurement but had no effect on the Ca⁺⁺ measurement; (2) the optimal conditions for specimen storage were no exposure to air at 4°C for up to 24 h; (3) no significant difference in the results of the Ca⁺⁺ concentration determined using AVL988/4 or i-STAT; (4) no carryover between samples was found.     

Calcium, Magnesium, and Phosphorus: Emergency Department Testing Yield

Objectives: To investigate how often the ED ordering of stat serum calcium (Ca⁺²), magnesium (Mg⁺²), and phosphorus (PO₄^-3) levels affected clinical treatment; to define the diagnoses of patients for whom Ca⁺², Mg⁺², and PO₄^-3 measurements did affect clinical therapy; and to suggest guidelines for more appropriate ordering of these laboratory tests. Methods: A retrospective chart review was performed in an academic teaching hospital. All adult ED patients who had Ca⁺², Mg⁺², or PO₄^-3 laboratory testing during the 9-month study period were included and evaluated for potential clinical impact of an abnormal Ca⁺², Mg⁺², or PO₄^-3 laboratory test. Results: 1,477 patients had Ca⁺², Mg⁺², or PO₄^-3 measured while in the ED during the study period. Of these, 260 patients (17.6%) had a total of 312 abnormal Ca⁺², Mg⁺², or PO₄^-3 values as defined by results exceeding ±15% of normal reference values. Of these, only 5 patients (0.3%) received treatment for abnormal values in the ED, while 75 patients (5.1%) were treated once admitted to the hospital. In this study, the only diagnostic groups to whom significant treatment was administered were diabetic patients (Ca⁺² and PO₄^-3); alcoholic patients (Mg⁺²); and renal failure patients (Ca⁺², Mg⁺², and PO₄^-3). Conclusion: These results suggest that stat Ca⁺², Mg⁺², and PO₄^-3 levels seldom affect clinical treatment in the ED. The frequency of ordering these tests may be reduced by obtaining Ca⁺², Mg⁺², or PO₄^-3 measurements only for patients known to be at risk for such abnormalities, based on their existing or suspected diagnoses. The authors suggest obtaining these tests, when indicated, on a “non-stat” basis, with the subsequent laboratory results becoming available in-hospital, where treatment is more likely to occur.     

Hypercalcitoninemia and inappropriate calciuria in the acute trauma patient

This study was undertaken to determine the role of calcium-regulatory hormones (calcitonin [CT], parathyroid hormone [PTH], and vitamin D analogs) during the first 48 hours after acute trauma. Eleven acutely traumatized patients admitted to the shock-trauma intensive care unit (STICU) in a tertiary care teaching hospital were enrolled. Eleven same-day elective surgery patients served as the control group. Levels of ionized calcium (Ca²⁺), total calcium, magnesium, phosphate, CT, PTH, vitamin D analogs, electrolyte supplementation, and renal electrolyte loss were recorded during the first 48 hours after admission to the STICU. Control-group measurements consisted of Ca²⁺ and CT. At admission, 91 % of the patients had ionized hypocalcemia (1.04 ± 0.10 mmol/L). Ca²⁺ levels increased significantly overtime (1.13 ± 0.08 at 24 hours; 1.16 ± 0.07 at 48 hours) but remained below the control-group value (1.28 ± 0.05; P < .05) despite supplementation. Ninety-one percent of the patients had increased CT values at admission, 91% at 24 hours, and 78% at 48 hours. Median CT values in the trauma patients were higher throughout the study than in the control group (P < .05). Urinary calcium loss in the trauma patients was within the normal range. PTH and vitamin D analog values were within the normal range throughout the study. Multiple regression analysis did not show any significant correlation between electrolytes and hormone or protein concentrations. Acute trauma patients have ionized hypocalcemia associated with inappropriate urinary calcium loss, increased CT levels, and normal PTH and vitamin D analog values. We believe the degree of calciuria we observed was inappropriate in the context of ionized hypocalcemia. The cause of these increased CT levels is unclear. Our results suggest that Ca²⁺-regulatory mechanisms may be disrupted in the acute trauma patient.     

Optimization of infusional calcium gluconate for prevention of hypocalcemic reactions during therapeutic plasma exchange

We sought to optimize direct intravenous infusion of calcium gluconate (CaGlu) for maintaining plasma ionized calcium concentration ([Ca²⁺]) and preventing hypocalcemic reactions during 34 consecutive 1-volume therapeutic plasma exchanges (TPEs) in eight patients. CaGlu, 2 g in 50 mL of 0.9% NaCl, was prepared by our hospital pharmacy and infused at either 1.0 or 1.6 g/h during alternate TPE. Plasma [Ca²⁺] was monitored at intervals of 20 to 30 minutes. At 1 g/h of CaGlu, plasma [Ca²⁺] fell by 8.35% after 40 to 50 minutes and then plateaued. At 1.6 g/h of CaGlu, plasma [Ca²⁺] fell by 6% after 20 to 30 minutes and then plateaued. The difference at 40 to 50 minutes was significant (P = .015). Hypocalcemic reactions were noted in three patients during 5 of 17 TPE at 1.0 g/h (all after 40 to 60 minutes) but 0 of 17 TPE at 1.6 g/h (P = .044). CaGlu at 1.6 g/h stabilized plasma [Ca²⁺] and appears to prevent hypocalcemic reactions during TPE.     

Serum magnesium level as a predictor of acute kidney injury in patients with acute pancreatitis

BACKGROUNDDecreased serum magnesium (Mg²⁺) is commonly seen in critically ill patients. Hypomagnesemia is significantly more frequent in patients with severe acute pancreatitis. Acute kidney injury (AKI) in patients with acute pancreatitis (AP) is associated with an extremely high mortality. The association underlying serum Mg²⁺ and AKI in AP has not been elucidated.AIMTo explore the association between serum Mg²⁺ on admission and AKI in patients with AP.METHODSA retrospective observational study was conducted in a cohort of patients (n = 233) with AP without any renal injury before admission to our center from August 2015 to February 2019. Demographic characteristics on admission, severity score, laboratory values and in-hospital mortality were compared between patients with and without AKI.RESULTSA total of 233 patients were included for analysis, including 85 with AKI. Compared to patients without AKI, serum Mg²⁺ level was significantly lower in patients with AKI at admission [OR = 6.070, 95%CI: 3.374-10.921, P < 0.001]. Multivariate logistic analysis showed that lower serum Mg²⁺ was an independent risk factor for AKI [OR = 8.47, 95%CI: 3.02-23.72, P < 0.001].CONCLUSIONOur analysis indicates that serum Mg²⁺ level at admission is independently associated with the development of AKI in patients with AP and may be a potential prognostic factor.     

Platelets oxidative stress in Indian patients with ischemic heart disease

Background: Oxidative stress has been implicated in the development of atherosclerosis and vascular tissue injury. Both platelet activation and lipid peroxidation are known to play major role in ischemic heart disease. The purpose of this study was to investigate the status of platelets oxidative stress in Indian patients with ischemic heart disease. Methods: We measured platelets aggregation, malonyldialdehyde (MDA), plasma‐ionized Ca²⁺, and antioxidant enzymes, i.e., glutathione peroxidase and superoxide dismutase in healthy volunteers and patients with myocardial infarction, unstable and stable angina 40 subjects each. Results: Platelets aggregation, MDA, and plasma‐ionized Ca²⁺ have increased significantly across the patients groups compared with controls, this increase was accompanied by an overall decrease in the antioxidant enzymes activity; except for the slight increases in the glutathione peroxidase levels among the myocardial infarction patients. Conclusions: The current results suggest that platelet lipid peroxidation as marked by increased MDA level is augmented in ischemic heart diseases. The increased oxidative stress seen in these patients was accompanied by platelet activation and impaired antioxidant enzymes activity. J. Clin. Lab. Anal. 24:49–54, 2010. © 2010 Wiley‐Liss, Inc.     

(Mg+ Ca-ATPase activity in plasma membrane enriched preparations of human skin fibroblasts: Decreased activity in fibroblasts derived from cystic fibrosis patients

Plasma membrane enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density centrifugation techniques were found to contain a Mg²⁺-dependent Ca²⁺-stimulated ATPase activity. The specific activity of the (Mg²⁺ + Ca²⁺-ATPase present was 4–5-fold higher than that present in crude membrane preparations and 80–100-fold higher than that present in homogenates. The (Mg²⁺ + Ca²⁺-ATPase activity of both crude and plasma membrane enriched preparations of cultured fibroblasts from cystic fibrosis patients was significantly reduced compared to that activity observed in age-matched controls. This study corroborates our previous observations made in crude homogenate preparations of fibroblasts and indicates another cell type where (Mg²⁺ + Ca²⁺-ATPase activity may be altered in cystic fibrosis.     

Effect of acute bile acid pool depletion on total and ionized calcium concentrations in human bile*

Abstract. Although calcium salts are important components of gallstones, there are few data on the total and ionized calcium content of human bile. Therefore, in 14 fasting T-tube patients studied 7–11 days after cholecystectomy, we measured bile flow, bile acid [BA], total [Ca_TOt] and free ionized [Ca⁺⁺] calcium concentrations, in 20–30 min bile collections during acute BA pool depletion induced by 6–8 h of continuous bile drainage. During washout of the BA pool there were parallel falls in bile flow, BA output and total calcium output (correlation coefficients ranging from 0.59 to 0.99; P< 0.02–0.001). In 12 of the 14 patients, [Ca_TOT] also fell (from 1.84 ± 0.29 to l.32 ± 0.34 mmol L^-1) in parallel with [BA] (from 34.0 ± 14.0 to 8.2 ± 8.0 mmol L^-1; r= 0.75–0.98; P<0.005). In contrast, biliary [Ca⁺⁺] remained virtually unchanged. These data suggest that the BAs are linked to the bound, rather than to the free, ionized, fraction of biliary calcium, which is consistent with in vivo calcium binding by BAs. A model is proposed in which BA-induced biliary calcium secretion results from (i) bile acid-induced water flow via solvent drag; and (ii) calcium binding in the bile canaliculus by bile acids, which induces paracellular diffusion of Ca⁺⁺, thereby maintaining [Ca⁺⁺] independent of [BA].     

The plasma membrane calcium ATPase modulates calcium homeostasis,intracellular signaling events and function in platelets

Summary. Background: The plasma membrane calcium ATPase (PMCA) regulates localized signaling events in a variety of cell types, although its functional role in platelets remains undefined. Objectives: To investigate the role of PMCA in determining platelet intracellular calcium concentration ([Ca²⁺]_i) at rest and following agonist stimulation, and to define the corresponding effects upon different stages of platelet activation. Methods: [Ca²⁺]_i was continuously measured in Fura‐2‐loaded platelets and in vitro and in vivo functional analyses performed in the presence of the PMCA inhibitor carboxyeosin (CE). Results: Concentrations of CE that selectively inhibited Ca²⁺ extrusion through PMCA were established in human platelets. [Ca²⁺]_i was elevated by CE in resting platelets, although collagen‐stimulated Ca²⁺ release was reduced. Impaired Ca²⁺ mobilization upon agonist stimulation was accompanied by reduced dense granule secretion and impaired platelet aggregation. Platelet aggregation responses were also reduced in PMCA4^?/? mice and in an in vivo mouse model of platelet thromboembolism. Conversely, inhibition of PMCA promoted the early and later stages of platelet activation, observed as enhanced adhesion to fibrinogen, and accelerated clot retraction. Investigations into the signaling mechanisms underlying CE‐mediated inhibition of platelet aggregation implicated cGMP‐independent vasodilator‐stimulated phosphoprotein phosphorylation. Conclusions: Disruption of PMCA activity perturbs platelet Ca²⁺ homeostasis and function in a time‐dependent manner, demonstrating that PMCA differentially regulates Ca²⁺‐dependent signaling events, and hence function, throughout the platelet activation process.     

Potential regulatory role of calsequestrin in platelet Ca2+ homeostasis and its association with platelet hyperactivity in diabetes mellitus

Summary. Background: Altered Ca²⁺ homeostasis contributes significantly to platelet hyperactivity in diabetes mellitus. Calsequestrin (CSQ), as a Ca²⁺ buffer protein in the sarcoplasmic reticulum, also regulates the Ca²⁺ release process in muscles. We hypothesized that CSQ may be expressed in platelets, but is altered and involved in diabetic platelet Ca²⁺ abnormalities and hyperaggregability. Methods: CSQ expression in platelets from streptozotocin‐induced type 1 diabetes rats, type 2 diabetes volunteers and Goto‐Kakizaki rats were analyzed by western blotting and RT‐qPCR. Platelet Ca²⁺ and aggregation were evaluated with Fura2 and an aggregometer, respectively. Results: Platelets from diabetic patients and rats exhibited increased resting Ca²⁺ levels, and hyperactive Ca²⁺ and aggregation responses to agonists. This enhanced basal Ca²⁺ was largely dependent on intracellular Ca²⁺ and insensitive to inositol 1,4,5‐trisphosphate receptor (IP₃R) antagonism. Additionally, the expression of the skeletal CSQ isotype (CSQ‐1) was detected in both rat and human platelets, but its levels were significantly lowered in diabetic platelets as compared with normal platelets. Impairment of CSQ by trifluoperazine caused concentration‐dependent Ca²⁺ release in normal platelets and HEK293 cells. Knocking down CSQ‐1 in HEK293 cells resulted in increased leakage of Ca²⁺, which was also insensitive to IP₃R inhibition, and exaggerated Ca²⁺ release following carbachol treatment. Conclusions: Downregulation of CSQ‐1 in diabetic platelets and impairment of CSQ‐1 in normal cells leads to disturbed Ca²⁺ release, demonstrating a potential role for CSQ‐1 in the regulation of the platelet Ca²⁺ release process and a possible causal contribution to diabetic platelet hyperactivity.     

Erythrocyte membrane properties in cystic fibrosis

The biochemical properties of the erythrocyte membrane have been studied in cystic fibrosis patients and in age-matched controls. No significant differences could be observed in either the concentration or composition of the principal membrane constituents. Contrary to previous reports, we found normal levels of the Mg²⁺-dependent and Ca²⁺ + Mg²⁺-dependent ATPase activities in CF preparations. However, a qualitative difference was observed in the Ca²⁺-dependent ATPase, since the enzyme had relatively greater activity at lower calcium concentrations in the cystic preparations. This effect on the membrane-bound Ca²⁺-ATPase is unlikely to represent a genetic defect in the enzyme itself but it may be connected with the mechanism of pathogenesis in cystic fibrosis.     

PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]i: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets

Summary. Background: The role of the tyrosine kinase pp60^src in PAR‐1‐dependent Ca²⁺ entry was investigated in human platelets. pp60^src plays a role in thapsigargin (TG)‐evoked store‐operated Ca²⁺ entry (SOCE), which is thought to be a major component of thrombin‐evoked Ca²⁺ entry. Methods: pp60^src tyr⁴¹⁶ phosphorylation was used to assess pp60^src activation. Fura‐2‐loaded platelets were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i). Results: Activation of PAR‐1 with the specific agonist SFLLRN increased pp60^src activation within 10 s. This required phospholipase C (PLC) activity, Ca²⁺ release and a rise in intracellular Ca²⁺. PP2, an inhibitor of Src‐family tyrosine kinases, inhibited SFLLRN‐evoked Ca²⁺ entry, but also inhibited Ca²⁺ release and the extrusion of Ca²⁺ by the plasma membrane Ca²⁺ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG‐ and SFLLRN‐evoked pp60^src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG‐evoked SOCE, it had little effect on SFLLRN‐ or thrombin‐evoked Ca²⁺ entry. Conclusions: These data indicate that stimulation of PAR‐1 leads to activation of pp60^src in human platelets, through PLC and cPKC activation, Ca²⁺ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN‐evoked Ca²⁺ entry, we suggest that pp60^src is also not required. The apparent inhibition of SFLLRN‐evoked Ca²⁺ entry by PP2 is likely to be secondary to reduced Ca²⁺ release. These data argue against a contribution of this SOCE pathway to PAR‐1‐dependent Ca²⁺ entry.     

Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion-exchange electrodes

Ion-exchange calcium electrodes represent the first practical method for the direct measurement of ionized calcium [Ca⁺⁺] in biologic fluids. Using both “static” and “flow-through” electrodes, serum [Ca⁺⁺] was within a rather narrow range: 0.94-1.33 mmoles/liter (mean, 1.14 mmoles/liter). Within a given individual, [Ca⁺⁺] varied only about 6% over a several month period. Consistent pH effects on [Ca⁺⁺] were observed in serum and whole blood, [Ca⁺⁺] varying inversely with pH. Less consistent pH effects were also noted in ultrafiltrates, believed to largely represent precipitation of certain calcium complexes from a supersaturated solution. Heparinized whole blood [Ca⁺⁺] was significantly less than in corresponding serum at normal blood pH, related to the formation of a calcium-heparin complex. [Ca⁺⁺] in ultrafiltrates represented a variable fraction (66.7-90.2%) of total diffusible calcium. There was no apparent correlation between serum ionized and total calcium concentrations. Thus, neither serum total calcium nor total ultrafiltrable calcium provided a reliable index of serum [Ca⁺⁺]. Change in serum total calcium was almost totally accounted for by corresponding change in protein-bound calcium [CaProt]. About 81% of [CaProt] was estimated to be bound to albumin and about 19% to globulins. From observed pH, serum protein, and [CaProt] data, a nomogram was developed for estimating [CaProt] without ultrafiltration. Data presented elsewhere indicate that calcium binding by serum proteins obeys the mass-law equation for a monoligand association. This was indicated in the present studies by a close correspondence of observed serum [Ca⁺⁺] values with those predicted by the McLean-Hastings nomogram. While these electrodes allow study of numerous problems not possible previously, they have not been perfected to the same degree of reliability obtainable with current pH electrodes. The commercial (Orion flow-through) electrode is: (a) expensive. (b) requires periodic replacement of membranes, and (c) has not yet been thermostated. As with blood pH measurements. (d) electrode response is logarithmic, i.e. small potential errors generate rather large [Ca⁺⁺] errors. (e) loss of CO₂ should be prevented, and (f) errors due to other cations must be considered under certain conditions. Despite these limitations, we believe the electrode represents a major advance in calcium metabolism.     

Monitoring the intracellular store Ca2+ concentration in agonist‐stimulated,intact human platelets by using Fluo‐5N

Summary. Background: Most Ca²⁺ signaling research in platelets has relied solely on monitoring the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). Changes in [Ca²⁺]_cyt constitute the net effect of Ca²⁺ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca²⁺ sequestration into the stores and Ca²⁺ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca²⁺ signaling difficult and subject to error. Objectives: To validate the use of the low‐affinity Ca²⁺ indicator Fluo‐5N to monitor the concentration of Ca²⁺ in the intracellular stores ([Ca²⁺]_st) of human platelets as a first step in developing assays for a systems‐level analysis of platelet Ca²⁺ signaling. Methods: Fluo‐5N‐loaded and Fura‐2‐loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca²⁺]_cyt and [Ca²⁺]_st. Results: Fluo‐5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca²⁺ stores and to physiologic agonists. Ca²⁺ reuptake inhibitors and blockers of Ca²⁺ release channels had the expected effects on Fura‐2 and Fluo‐5N fluorescence. Agonist‐evoked Ca²⁺ release was reversed by Ca²⁺ addition to the medium, and required intact Ca²⁺ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non‐SERCA Ca²⁺ reuptake mechanism. Evidence for a role for Ca²⁺‐induced Ca²⁺ release in agonist‐evoked responses was obtained. Conclusions: Our data provide a validation of the use of Fluo‐5N as a method for monitoring changes in [Ca²⁺]_st in human platelets.     

Cytoplasmic calcium regulation and the electrocardiogram in patients with primary hyperparathyroidism

Summary. Primary hyperparathyroidism (HPT) is a disease caused by an abnormal cytosolic regulation of calcium concentration [Ca⁺⁺]_i leading to an increased secretion of parathyroid hormone and thereby increased levels of extracellular calcium. It is well known that the QT-interval measured at electrocardiography (ECG) is shortened in HPT subjects. Whether this is due to an abnormal intracellular handling of calcium also in the heart or to the raised extracellular calcium levels is not known. In order to study the extent to which the deranged extra- and intracellular levels of calcium in HPT patients were related to ECG characteristics, [Ca⁺⁺]_i was determined in vitro by microfluorometry in surgery-removed parathyroid cells at extracellular calcium concentrations of 0–5 mM and 30-0 mM and ECG was recorded preoperatively in 42 HPT patients and in 15 subjects operated on for atoxic goitre. Serum calcium and plasma-ionized calcium also were measured preoperatively. The QT-interval and ST-segment duration were both shortened in the HPT patients compared to controls (P < 0–001). [Ca⁺⁺]_i at 3-0 mM extracellular calcium divided by that at 0–5 mM was correlated to the QT-interval, when measured at the onset of the T-wave (QoT, r= 0–39, P < 0–03) and early diastolic phase (end of T-wave to onset of p-wave, r= -0–34, P= 0–05). Plasma ionized calcium was related inversely to the QT-interval, when measured to the apex of the T-wave (QaT, r= -0–48, P < 0–04), while the non-ionized form of calcium in serum was correlated to the length of the PQ-interval (r= 0–53, P< 002). The study described here showed the altered cytosolic calcium levels in the HPT subjects to be related to QoT-interval and early diastolic phase implying that a defect intracellular handling of calcium could be present also in the heart to cause these ECG changes. On the other hand, the levels of plasma ionized and serum non-ionized calcium were related to ECG characteristics suggesting that the extracellular levels of this ion are important.,     

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca²⁺ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9 : 1599–607. Summary. Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca²⁺ signaling must be tightly controlled. Thrombin‐induced Ca²⁺ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca²⁺signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin‐induced Ca²⁺ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca²⁺ signaling was monitored in fura‐2‐loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide‐I (BIM‐I) enhanced α‐thrombin‐induced Ca²⁺ signaling in a concentration‐dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐thrombin‐induced signaling following treatment with the PAR1 antagonist, SCH‐79797. BIM‐I enhanced Ca²⁺ release from intracellular stores and Ca²⁺ entry, as assessed by Mn²⁺ quench. However, the plasma membrane Ca²⁺ ATPase inhibitor, 5(6)‐carboxyeosin, did not prevent the effect of BIM‐I. PKC inhibition strongly enhanced α‐thrombin‐induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca²⁺ release and Ca²⁺ entry, which leads to reduced platelet PS exposure.