Calcium Ion Mobilization Research Articles

Preferential interaction of the CD4+29+/45RA-subset of human CD4+ T lymphocytes with an antibody against the cell-membrane ganglioside GD3.

This study shows that the two major subpopulations of CD4+ T lymphocytes defined on the basis of differential expression of the CD29 and CD45RA antigens, show significant differences in reactivity with a monoclonal antibody against the GD3 ganglioside. Double staining studies showed that the GD3 ganglioside is predominantly expressed on cells from the CD4+29+/45RA subsets. The preferential interaction of the CD4+29+/45RA cell subset with the anti-GD3 MoAb was further confirmed by the proliferative and calcium-flux studies. Accordingly, the reciprocal, CD4+(29-)/45RA+ subset was unable to proliferate in response to the anti-GD3 MoAb alone. Although it did show a significant mobilization of calcium ions and proliferation to IL-2 when stimulated with the anti-GD3 MoAb, these response were much less pronounced than the responses of the CD4+29+/45RA- subset. Finally, when two T-cell stimulating monokines, IL-1 and IL-6, were tested for the ability to modulate the anti-GD3 mediated proliferation, only the former, but not the latter was able to enhance the proliferation. Although the natural ligand for the GD3 ganglioside remains unknown, the data presented here provide further evidence in support of the notion that the T-cell surface molecules different from the T-cell receptor MHC-antigen complex may contribute to the preferential activation of one of the CD4+ T lymphocyte subsets.

Effects of the prostanoid receptor antagonist, di-4-phloretin phosphate, upon human sperm motility

The inhibitory effects of the prostanoid receptor antagonist, di-4-phloretin phosphate (DPP), upon washed human sperm motility were determined in vitro (up to 60 min of incubation). Concentrations tested were: 0.001, 0.01, 0.1, 0.25, 0.5, 2.5, 5.0 and 10.0mM. All concentrations of DPP investigated caused cessation of sperm movement (percentage motility and average forward velocity) and the antimotility effect was essentially irreversible. The concentrations producing 50% inhibition were: percentage motility −1.31 ± 0.56mM (mean ± SEM) and average forward velocity −1.95 ± 0.68mM. The mode of antimotility action appears to be multi-faceted. At high (10mM) and intermediate (2.5mM) concentrations, changes in fluidity of sperm plasmalemma (hypoosmotic swelling test) and loss of viability (nigrosin-eosin stain technique) constitute the primary means by which motility was disrupted. In contrast, these two parameters of motility remained unaltered at a lower concentration (0.25mM) of DPP, and the mechanism precipitating the antimotility effect may involve mobilization of stored calcium ions and modulation of cyclic AMP levels.

Mechanism of action of MY-1250, an active metabolite of Repirinast, in inhibiting histamine release from rat mast cells.

1. When MY-1250 (3.6 x 10(-5) M) was added to mast cells, it caused a rapid increase in adenosine 3':5'-cyclic monophosphate (cyclic AMP) and decrease in adenosine 5'-triphosphate (ATP), both of which recovered to their original levels within 2 min. The accumulation of cyclic AMP was maximal at 20 s after challenge with MY-1250. The minimum level of ATP was observed at 20 s after addition of MY-1250. 2. The initial rise in [Ca2+]i and the histamine release induced by DNP-AS antigen (40 micrograms ml-1) was most strongly inhibited at 20 s after incubation of the mast cells with MY-1250. 3. MY-1250 strongly and dose-dependently inhibited the histamine release from rat mast cells induced by antigen. Moreover, MY-125 strongly inhibited calcium ion mobilization from the intracellular Ca(2+)-store. 4. These results suggested that the inhibitory mechanism of MY-1250 on the initial rise in [Ca2+]i and histamine release induced by antigen was due to the inhibition of ATP-dependent Ca(2+)-release from the intracellular Ca(2+)-stores.

Evaluation of platelet calcium ion mobilization by the use of various divalent ions

Divalent ion mobilization in human platelets was evaluated with Fura-2 fluorescence changes induced by Ca 2+, Sr 2+, Ba 2+ and Mn 2+. Extracellular Ca 2+, Sr 2+ and Ba 2+ all entered thrombin-stimulated platelets. These divalent ions were also able to refill the intracellular Ca 2+ storage sites which had been depleted of Ca 2+ by ionomycin treatment, and were released from the storage sites upon thrombin stimulation. However, only the refilling of the storage sites with Ca 2+ and Sr 2+, but not with Ba 2+, were capable of suppressing the opening state of Ca 2+ channels assessed with Mn 2+ influx. Efflux of intracellularly accumulated divalent ions was observed with Ca 2+ and Sr 2+ but not with Ba 2+. These findings indicate that there are subtle differences in the Ca 2+-binding domains of the various systems involved in Ca 2+ mobilization in platelets, some of which discriminate Ba 2+ while accepting Sr 2+.

Calcium ion mobilization in neuronal cells induced by PAF

We have reported previously that platelet-activating factor (PAF) interacts with the neuronal cell line NG108-15 (neuroblastoma X glioma hybrid) and the pheochromocytoma cell line, PC12. PAF acts on these cells by raising levels of intracellular free calcium ions. In the present report, we extend these studies. PAF induced the vesicular release of adenosine 5'-triphosphate (ATP) from PC12 cells in a dose-dependent manner. The PAF-induced ATP release was inhibited by the PAF antagonists, CV-3988 and CV-6209, and the calcium antagonist prenylamine. The relevance of the interaction of PAF with neuronal cells was investigated further by using brain synaptosomal preparations and primary cortical and neostriatal cells. Nanomolar concentrations of PAF induced calcium transients in aequorin-loaded synaptosomal preparations, and cortical and neostriatal cells were sensitive to the action of PAF. The possible physiological and pathophysiological roles of PAF in brain function are discussed.

Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.

Caffeine-induced contractions in rabbit isolated renal artery are differentially inhibited by calcium antagonists

Caffeine (1–60 mM) induced concentration-dependent, endothelium-independent phasic contractile responses in isolated rabbit renal artery ring preparations. For concentrations of caffeine over 2 mM, responses were mainly the result of intracellular calcium ion mobilization since they were relatively resistant to removal of calcium ions from the bathing medium. The L-type slow calcium channel blocker, nifedipine (10 μM), had no effect and high concentrations of verapamil and diltiazem (10–30 μM) only slight and inconsistent effects (not concentration-dependent) upon these caffeine responses. Likewise, the highly lipophilic calcium antagonists flunarizine and lidoflazine (3–30 μM) only slightly displaced caffeine concentration-response curves to the right and reduced the maximum response. These small inhibitory effects of flunarizine and lidoflazine were not augmented in a calcium-free medium. In contrast, the other lipophilic calcium antagonists, bepridil and fendiline (3–30 μM), produced marked, non-competitive type inhibition of caffeine responses, completely inhibiting responses to the alkaloid at the highest concentration. Furthermore, the inhibitory effects of bepridil and fendiline were markedly augmented in calcium-free medium. These results clearly differentiate bepridil and fendiline from the other calcium antagonists studied. In addition they provide further evidence for effects other than at the cell membrane which could theoretically contribute to the efficacy of bepridil and fendiline as anti-anginal agents.

Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing the superoxide (O2-) generation of human neutrophils, but it enhanced the O2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) about 2-fold when the cells had been preincubated with ET-1 for 10 min at 37 degrees C. The concentration at which ET-1 was 50% effective was 1 x 10(-10) M, and the maximal effect was obtained at 1 x 10(-8) M. The enhancement was observed over the range of the effective concentrations of FMLP (10(-8)-10(-6) M). ET-1 did not promote the mobilization of intracellular calcium ions and the enhancing effect of ET-1 did not change when calcium ions were depleted. These findings indicate that ET-1 is a potent modulator of human neutrophils and may thus contribute to the inflammatory process.

Inhibitory effects of MK‐886 on arachidonic acid metabolism in human phagocytes

1. We have investigated the inhibitory activity of compound MK-886 (formerly L-663,536), an indole derivative, on 5-lipoxygenase product synthesis in various human phagocytes stimulated with either the ionophore A23187, in the presence and absence of exogenous arachidonic acid, or platelet-activating factor (PAF). The lipoxygenase products were analysed by reversed-phase h.p.l.c. 2. MK-886 inhibited the formation of 5-hydroxy-eicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), its omega-oxidation products and 6-trans-isomers with an IC50 value of 10-14 nM in A23187-stimulated neutrophils. In the same system, nordihydroguaiaretic acid (NDGA), AA-861 and L-655,240 showed IC50 values of 250-510, 110-420 nM and 1.7-3.9 microM, respectively. 3. MK-886 inhibited 5-lipoxygenase product synthesis in A23187-stimulated blood eosinophils and monocytes, and in neutrophils primed with granulocyte-macrophage colony-stimulating factor and stimulated with PAF with IC50 values of 1-13 nM. 4. The inhibitory activity of MK-886 was not reversed by addition of 10 microM arachidonic acid to A23187-stimulated neutrophils. 5. Compound MK-886 had no effect on 15-lipoxygenase product synthesis in blood eosinophils and neutrophils up to a concentration of 1 microM. 6. At 100 nM compound MK-886 had no significant effects on calcium ion mobilization, superoxide anion production and actin polymerization in neutrophils. 7. In conclusion, MK-886 is a very potent and specific inhibitor of both LTB4 and LTC4 synthesis in various types of human phagocytes.

Structure-Function Relationships for Full-Length Recombinant Parathyroid Hormone-Related Peptide and Its Amino-Terminal Fragments: Effects on Cytosolic Calcium Ion Mobilization and Adenylate Cyclase Activation in Rat Osteoblast-Like Cells*

PTH-related peptide (PTHrP) may be a major cause of the humoral hypercalcemia of malignancy. The circulating form of PTHrP is unknown, but mRNA analysis of tumor tissue suggests that multiple forms of PTHrP may exist. Therefore, we examined the ability of the full 141-amino acid protein as well as 2 amino-terminal fragments, PTHrP-(1-34) and PTHrP-(1-74), to increase cytosolic calcium ion concentrations ([Ca2+]i; assessed by aequorin luminescence) and stimulate cAMP accumulation in osteoblast-like rat osteosarcoma cells (ROS 17/2.8). PTH and all PTH-related peptides examined increased [Ca2+]i and cAMP in a concentration-dependent manner. The [Ca2+]i response to PTHrP-(1-34) closely resembled that to rat PTH-(1-34); both peptides produced biphasic responses. However, the responses to the longer PTHrP fragments generally were not biphasic. There were no significant differences among the three PTHrP forms in increasing [Ca2+]i or stimulating cAMP accumulation, although PTHrP-(1-74) was consistently weaker than the other two PTHrP peptides. PTHrP-(1-34) was more potent than rPTH-(1-34), which, in turn, was more potent than human PTH-(1-34) in increasing [Ca2+]i. However, PTHrP-(1-34) was not consistently more potent than either human PTH-(1-34) or rat PTH-(1-34) in stimulating cAMP accumulation. The inhibitory PTH analog bovine PTH-(3-34) attenuated both cAMP and [Ca2+]i responses to PTHrP-(1-34), but bovine PTH-(7-34) only reduced the [Ca2+]i response. Our data are generally consistent with PTHrP's acting through the PTH receptor, but differences in the effects of inhibitory PTH analogs on PTH and PTHrP action suggest as yet unexplained complexities, such as the existence of a PTH/PTHrP receptor family.

Inhibition of Histamine Release from Rat Peritoneal Mast Cells by MY-1250, an Active Metabolite of Repirinast (MY-5116)

MY-1250, an active metabolite of Repirinast (MY-5116), strongly and dose-dependently inhibited the in vitro histamine release from rat peritoneal mast cells induced by antigen. The IC50 value of MY-1250 for histamine release was in the order of 0.3 microM. Moreover MY-1250 strongly inhibited calcium ion mobilization from the intracellular Ca2+ store. In the presence of 3.6 x 10(-5) M of MY-1250, the initial rise in [Ca2+]i reached a maximum of only 13 nM, and the inhibitory effect was 81% for the initial rise. The IC50 value for this event was 0.25 microM. The inhibitory effect of MY-1250 on the initial rise in [Ca2+]i was closely correlated with that on histamine release.

Lipoxin A4 and Lipoxin B4 Inhibit Chemotactic Responses of Human Neutrophils Stimulated by Leukotriene B4 and N-Formyl-l-Methionyl-l-Leucyl-l-Phenylalanine

1. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) have been evaluated for their capacities to modulate neutrophil (PMN) migration and endothelial cell adherence using compounds prepared by total chemical synthesis. 2. Increased PMN migration was seen with concentrations of LXA4 from 10(-9) mol/l to 10(-7) mol/l. LXA4 was 100-fold less potent than leukotriene B4 (LTB4) and it elicited only one-half of the maximal response of LTB4. 3. The (5S,6S,15S)-isomer of LXA4 induced only a weak migratory response and LXB4 was inactive, suggesting that the activity of LXA4 was stereospecific. 4. Modified chequerboard analysis indicated that LXA4 was a chemokinetic agent. 5. Preincubation of PMN with increasing concentrations of LXA4 induced a very similar dose- and time-dependent inhibition of the subsequent response to 10(-7) mol/l LTB4 or 10(-7) mol/l N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). The inhibition was observed at 10(-10) mol/l LXA4; the concentration which produced 50% inhibition was 10(-8) mol/l and 100% inhibition of PMN locomotion occurred at 10(-6) mol/l LXA4. 6. The (5S,6S,15S)-isomers of LXA4 and LXB4 were 5- and 100-fold less potent than LXA4, respectively, in suppressing LTB4- or FMLP-induced PMN migration. 7. Preincubation of PMN with LXA4 led to a suppression of calcium mobilization, as assessed by Quin2-AM fluorescence, when the cells were subsequently stimulated under optimal conditions by LTB4 or FMLP. 8. These results suggest that the inhibitory activity of lipoxins may be related to the capacity of these molecules to regulate calcium ion mobilization.

Development of a Novel Biocompatibility Test Method Based on Intracellular Calcium Ion Mobilization

Development of a Novel Biocompatibility Test Method Based on Intracellular Calcium Ion Mobilization

Development of a Novel Biocompatibility Test Method Based on Intracellular Calcium Ion Mobilization

This paper presents a novel evaluation method for determining platelet/material interaction at the transmembrane level by incorporation of a Ca-sensitive fluorophore (Fura-2) into platelets. The transmembrane stimulation of platelets which are eluted from commercially available artificial dialyzers was quantitatively measured as the elevation of intracellular free calcium ion concentration. This method clearly differentiated platelet response to hydrophilic (ethylene vinyl alcohol copolymer and cellulose) vs. hydrophobic (polyacrylonitrile and cellulose acetate) membranes, and offers a versatile means to understanding platelet/material interaction.

Nicorandil: Animal pharmacology

Nicorandil, a nicotinamide derivative, is a recently developed, orally active antianginal drug with a cardioprotective activity, and its pharmacologic properties differ from those of conventional antianginal drugs. Nicorandil has the capacity to increase myocardial oxygen supply without increasing oxygen demand by reduction in preload and afterload. In isolated blood-perfused canine heart preparations, when injected into the sinus node, the atrioventricular node or the anterior septal arteries, nicorandil at dose levels doubling blood flow through the respective arteries has virtually no effect on sinus rate, atrioventricular conduction time or contractile force of ventricular muscle. This may indicate that nicorandil possesses a selective effect on the coronary vasculature rather than on the myocardium. Furthermore, the vasospasmolytic activity of nicorandil has been evidenced in in vivo and in vitro experiments. The precise mechanism by which nicorandil develops coronary vasodilating and vasospasmolytic effects remains to be elucidated, but it may be partly inferred by an increase in the potassium conductance in the membrane, a relation with cyclic guanosine monophosphate formation, or inhibition of intracellular calcium ion mobilization in the cell of coronary vascular smooth muscle. Nicorandil possesses a nitrate moiety in its chemical structure. However, it is noted that nicorandil unlike nitrates does not develop tolerance or cross tolerance to other conventional nitrates in terms of blood-pressure lowering effects and coronary vasodilating effects. Thus, nicorandil is likely to have highly beneficial properties in the treatment of angina pectoris.

Regulation of relaxin release from monodispersed porcine luteal cells: effect of calcium ionophore A23187 and calcium channel blockers.

The role of calcium ion mobilization in modulation of secretion of the ovarian protein hormone relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells. The rate of development of plaques in time-course experiments was used in this study as an index of the rate of relaxin release. Exposure of luteal cell-containing monolayers to the calcium-mobilizing agent A23187 (40 nM to 5 microM) resulted in a dose-related increase in the rate of relaxin-plaque formation. This effect [and the influence of a stimulatory secretagogue, prostaglandin E2 (PGE2; 1 microM)] was suppressed by coculture with Co2+ (5 mM), a calcium channel blocker. These results are consistent with the view that calcium ion redistribution within porcine luteal cells forms a pathway that subserves, at least in part, the rates of basal and stimulated relaxin release in vitro. However, A23187 was equally effective in enhancing the rate of plaque formation when the monolayers were bathed in a low calcium medium (mean +/- SEM, 6.61 +/- 0.92 microM Ca2+), rather than a calcium-replete medium (1.56 +/- 0.09 mM Ca2+). Likewise, neither basal nor PGE2-stimulated (1 microM) relaxin secretion was abrogated by culture of monolayers in low calcium medium. These data suggest that the stimulatory effect of A23187 (and perhaps PGE2) arose predominantly through redistribution of calcium stored within intracellular sites in luteal cells, rather than entrance of calcium into the cell from the extracellular medium. Yet, incompatible with this interpretation, we observed that TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; a putative inhibitor of intracellular calcium redistribution] enhanced rather than blocked A23187- or PGE2-stimulated relaxin release. This result is consistent with the possibility that TMB-8 mobilized (rather than blocked) calcium in this cell system or that it acted via calcium-independent mechanisms. We conclude that calcium mobilization has the potential to act as a molecular pathway that transduces secretion of an ovarian peptide hormone, relaxin. However, the exact nature and physiological role(s) of the Ca2+ pathway in the control of relaxin secretion and the interrelationships of this mechanism with other intracellular messengers that may also modulate ovarian peptide secretion remain to be more clearly defined.

Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells.

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.

Angiotensin II induces expression of the [formula omitted] gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c- fos mRNA level. The doses of this agonist necessary for the increase in the c- fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12- O -tetradecanoylphorbol-13-acetate and Ca 2+-ionophore A23187 increased the c- fos mRNA level in an additive manner. These results suggest that angiotensin II induces enpression of the c- fos gene through the activation of protein kinase C and Ca 2+ mobilization in cultured vascular smooth muscle cells.

Regulation of receptors on parietal cells on acid secretion.

Four types of receptors have so far been identified on the parietal cell: receptors for gastrin, acetylcholine, and histamine, whose activation leads to stimulation of acid secretion, and receptors for prostaglandins of the E series, whose activation leads to inhibition of acid secretion. Postreceptor events following drug-receptor interaction include mobilization of calcium ions and cAMP formation. Moreover, a number of putative receptors (for somatostatin, CCK, adenosine, secretin, etc.) have been hypothesized, even though definite evidence is still lacking. Finally, other substances may influence gastric acid secretion in a positive or negative way, but no evidence for an involvement of specific receptors on the parietal cells is available so far.

The effects of plasma volume variations on the calcium concentration during epidural anaesthesia.

The plasma variations of the ionized and total calcium concentration in relation to protein concentration and plasma volume were studied in 12 patients during epidural anaesthesia for transurethral prostatectomy. The most significant changes were noted during the onset of anaesthesia. From arrival in the operating theatre to start of operation, the plasma concentration of ionized calcium decreased from 1.28 (s.d. = 0.05) mmol/l to 1.22 (s.d. = 0.04) mmol/l and total calcium decreased from 2.39 (s.d. = 0.11) mmol/l to 2.17 (s.d. = 0.10) mmol/l. At the same time, however, the decrease in ionized calcium concentration and the increase in plasma volume were associated with a rise of 10.3% in the pool of free calcium ion in plasma. After the start of surgery, the plasma ionized calcium concentration remained almost unaffected throughout the rest of the study period. The total plasma calcium concentration as well as the total protein concentration remained stable during operation, only increasing after the end of surgery. The preoperative changes in the plasma calcium concentration may have been the result of both haemodilution and mobilization of calcium ions from its bone stores. Postoperatively, an increased urinary loss of calcium must have followed the administration of furosemide at the end of surgery.