Imaging of Ca2+-induced cytoplasmic Ca2+ responses in normal and pathological parathyroid cells

Ca²⁺-induced changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) were studied in bovine and normal and pathological human parathyroid cells using digital image analysis of fura-2-loaded cells. When raising external Ca²⁺ from 0.5 to 3.0 mmol L⁻¹, about 95% of all cells reacted rapidly and simultaneously with sustained elevation of [Ca²⁺]_i. In approximately two out of three bovine parathyroid cells, normal human cells and cells from most patients with hyperparathyroidism (HPT) the sustained phase was preceded by an overshooting [Ca²⁺]_i transient. The proportion of cells with such a transient was decreased in cells from severe cases of uraemic parathyroid hyperplasia only. However, pathological human cells from adenomas and normal-sized glands associated with adenomas, as well as cells from primary and uraemic hyperplasias, had lower peak and sustained levels than normal human and bovine cells. The results indicate that both normal and pathological parathyroid cells exhibit heterogeneity in their [Ca²⁺]_i responses to elevation of external Ca²⁺. The Ca²⁺-induced [Ca²⁺]_i transients and the sustained elevations are attenuated in pathological human parathyroid cells. However, the presence of the overshooting transient represents physiological variability rather than being a consequence of the pathophysiology associated with HPT.     

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery

Summary— The involvement of large conductance Ca²⁺-activated K⁺ channels (BK) and ATP-sensitive K⁺ (K_ATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca²⁺]_i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca²⁺]_i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca²⁺]_i with force in the presence of serotonin at different extracellular Ca²⁺ concentration ([Ca²⁺]_o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca²⁺-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.     

Inhibitory effects of okadaic acid on rat uterine contractile responses to different spasmogens

Summary— In the present study, we examined the effects of okadaic acid, a selective inhibitor of type 1 and 2A protein phosphatases, on the mechanical responses evoked by oxytocin, K⁺- and Na+-modified solutions and ouabain in estrogen-primed rat myometrium. Oxytocin elicited a rapid, phasic contraction followed by rhythmic oscillations. The phasic response was partially resistant to the absence of external Ca²⁺. Okadaic acid (1 μM) and the L-type calcium channel blocker nifedipine (1 μM) abolished the oscillatory component and reduced the initial, phasic response to about 80% of the control response. High K⁺ (60 mM) solution, ouabain (1 mM), K⁺-free medium and low Na⁺ (25 mM) solution induced extracellular Ca²⁺-dependent biphasic responses composed by an early rapid (KCl, ouabain and K⁺-free solution) or slower developed (25 mM Na⁺ solution) phasic contraction followed by a sustained increase in tension. Okadaic acid and nifedipine, alone or in combination, abolished or decreased similarly the contractile response evoked by these stimulants. The okadaic acid- and nifedipine-insensitive responses to ouabain, K⁺-free and low Na⁺ solution were enhanced by increasing the extracellular concentration of Ca²⁺ in the medium and were inhibited in a dose-dependent manner by amiloride (0.05–0.5 mM). These data suggest that, in estrogen-primed rat uterus, dephosphorylating mechanisms by OA-sensitive protein phosphatases play an important role in regulating myometrial contractions elicited by Ca²⁺ entry through voltage-sensitive Ca²⁺ channels.     

Interactions of calcium, magnesium and atropine on exocrine pancreatic secretion in man

Abstract. The effects of the intravenous administration of atropine or magnesium on pancreatic secretion which has been stimulated by secretin and induced hypercal-caemia have been studied in man.
In the presence of secretin (0.5 CU/kg.h) the infusion of Ca²⁺ (0.3 mmol/kg.105 min) resulted in an increase in secretion of enzymes by 100–200%, and in that of Ca²⁺ and Mg²⁺ by 50–100% without affecting fluid and bicarbonate secretion.
The additional injection of atropine (0.5 mg i.v. and 0.5 mg s.c.) were followed by a prompt fall in enzymes but not in Ca²⁺ and Mg²⁺ to the secretin-stimulated values.
The additional infusion of Mg²⁺ (0.12 mmol/kg.45 min) to the Ca²⁺-infusion did not alter the secretion of enzymes, Ca²⁺ or Mg²⁺ compared with the calcium infusion alone.
It is suggested that the hypercalcaemic stimulus depends on an intact innervation of the acinar cells.
In these experiments the secretion of Ca²⁺ and Mg²⁺ seem to originate mainly from extracellular fluxes.     

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells

Summary— The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K⁺, and on voltage-dependent Na⁺ and Ca²⁺ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 μM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 μM) did not. In dispersed cells, both tacrine (1 μM-1 mM) and physostigmine (1 μM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K⁺ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1–100 μM) or physostigmine (1 μM-1 mM). In fura-2 loaded cells, tacrine (100 μM) almost abolished [Ca²⁺]i rise induced by veratridine, but only slightly reduced that evoked by high K⁺. In voltage-clamped cells, tacrine (300 μM) depressed the voltage-dependent Na⁺ and Ca²⁺ currents by about 93% and 69%, and physostigmine (300 μM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na⁺ channels rather than the Ca²⁺ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.     

Calcium signaling in platelets

Summary.  Agonist-induced elevation in cytosolic Ca²⁺ concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca²⁺ release from intracellular stores and Ca²⁺ entry through the plasma membrane (PM). Ca²⁺ store release is a well-established process involving phospholipase (PL)C-mediated production of inositol-1,4,5-trisphosphate (IP₃), which in turn releases Ca²⁺ from the intracellular stores through IP₃ receptor channels. In contrast, the mechanisms controlling Ca²⁺ entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, as in other non-excitable cells, the major way of Ca²⁺ entry involves the agonist-induced release of cytosolic sequestered Ca²⁺ followed by Ca²⁺ influx through the PM, a process referred to as store-operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca²⁺ entry mechanism is mediated by the direct receptor-operated calcium (ROC) channel, P2X₁. Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca²⁺ entry through the PM. This review summarizes the current knowledge of platelet Ca²⁺ homeostasis with a focus on the newly identified Ca²⁺ entry mechanisms.     

Hypoxic contractile response in isolated rat thoracic aorta: role of endothelium, extracellular calcium and endothelin

Summary— The effects of hypoxia on isolated arteries remain controversial, depending on the species, vascular beds and protocols. The aims of the study were to characterize the response of rat thoracic aorta to hypoxia and to examine the roles of endothelium, extracellular calcium and endothelin in this response. Hypoxia was induced by bubbling Krebs solution with 95% N₂ and 5% CO₂ instead of 95% O₂ and 5% CO₂. Experiments were performed during 1 h in norepinephrine (0.01 μM) precontracted rings. Hypoxia produced a biphasic response consisting of an initial transient partial relaxation (67% at 14 min) followed by a slow but sustained contraction (27% from 40 to 60 min). After endothelium removal, relaxation appeared faster with increased magnitude (82% at 12 min) and was followed by a weak transient contraction (16% at 25 min). In endothelium-intact rings, Ca²⁺ free medium (EGTA, 0.1 mM) and Ca²⁺ channel blockers, verapamil (0.05, 0.5 and 5 μM) or nicardipine (0.1, 1 and 10 μM), had no effect on relaxation but inhibited the contraction, the effects of both calcium antagonists being concentration-dependent. Similarly, the ET_A/ET_B receptor antagonist, bosentan (0.1, 10 and 1,000 nM), induced a concentration-dependent decrease in the contraction. We conclude that 1) the response of rat thoracic aorta during 1 h of hypoxia is biphasic (relaxation followed by contraction); 2) the endothelium is involved in the contraction whereas its role in the relaxation remains to be elucidated; 3) extracellular calcium is involved in the contraction; and 4) endothelin may play a role in the contraction.     

Dynamics of the cationic and secretory responses to A-4166 in perifused pancreatic islets

Summary— The dynamics of the cationic and secretory response to A-4166, a hypoglycemic meglitinide analogue, was investigated in rat islets prelabelled with either ⁸⁶Rb or ⁴⁵Ca and placed in a perifusion system. In the absence of D-glucose, A-4166 (10 μM) provoked an immediate, sustained and rapidly reversible inhibition of ⁸⁶Rb outflow, this contrasting with a short-lived stimulation of insulin release. In the presence of 6 mM D-glucose, A-4166 provoked a rapid, sustained and rapidly reversible stimulation of both insulin release and ⁴⁵Ca efflux. The latter cationic response was suppressed in the absence of extracellular Ca²⁺, in which case A-4166 even caused a modest decrease in effluent radioactivity.
These findings support the view that A-4166 acts mainly in the islet B-cell by closing ATP-responsive K⁺ channels, leading to subsequent depolarization of the plasma membrane and gating of voltage-sensitive Ca²⁺ channels. Independently of the latter effect, A-4166 may also affect the intracellular distribution of Ca²⁺ ions. The present data further indicate that A-4166 belongs to those hypoglycemic agents that cause rapidly reversible changes in cationic and secretory events, at variance with highly potent sulfonylureas such as glibenclamide or glimepiride.     

The cytoplasmic Ca2+ response to glucose as an indicator of impairment of the pancreatic β-cell function

Abstract. The effects of glucose on the cytoplasmic Ca²⁺ concentration (Ca²⁺_i) regulating insulin release were investigated using pancreatic β-cells representative for the normal and diabetic situations. Increase of the glucose concentration resulted in a slight lowering of Ca²⁺_i followed by a rise, often manifested as high amplitude oscillations. The Ca²⁺_i-lowering component in the glucose action associated with suppression of insulin release became particularly prominent when the β-cells were already depolarized by tolbutamide. Glucose-induced inhibition of insulin release was observed also in experiments with rats made diabetic with streptozotocin or alloxan. Other studies indicated lowering of plasma insulin after intravenous glucose administration in patients with insulin- and noninsu-lin-dependent diabetes mellitus. Brief exposure of β-cells to 2–2 mmol 1^-1 streptozotocin resulted in impairment of the response to glucose, manifested as disappearance of the cyclic variation of Ca²⁺_i. The results indicate that glucose-induced depolarisation is a vulnerable process, the disturbance of which may contribute to insulin secretory defects in diabetes mellitus.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Comparative inotropic effect of calcium, endothelin-1, and forskolin on ferret papillary muscles during acidosis

Summary— Acidosis affects multiple steps in the excitation-contraction coupling pathway of myocardium, producing decreased calcium sensitivity of myofibrils and modification of the function of the sarcoplasmic reticulum. Our aim was to evaluate the effectiveness of three different classes of inotropic agents under acidotic conditions: 1) forskolin, an adenylate cyclase activator that enhances cellular cyclic AMP concentrations, 2) elevated extracellular Ca²⁺ and 3) endothelin-1, an activator of the inositol triphosphate, diacylglycerol pathway. Ferret papillary muscles were mounted in organ baths containing normal physiological solution (pH = 7.4). After baseline tension was measured, the muscles were bathed in an acidotic solution (pH = 6.98) that decreased tension to 40% of the control; subsequently, the muscles were washed with normal physiological solution until they returned to baseline. Each inotropic agent was added to the bathing solution in a concentration sufficient to increase tension by 40% above the baseline. Then the solution was made acidotic (pH = 6.98) in the continuous presence of that concentration of inotropic agent and the resultant steady-state developed tension measured. The increases in tension induced by each inotropic agent at normal pH were adjusted to be similar; in contrast, the response to each drug in acidosis was significantly different. Under acidotic conditions, endothelin-1 was the most effective inotropic agent in restoring the depressed developed tension. This was possibly due to enhancement of the myofilament sensitivity to Ca²⁺, which was more effective than increasing [Ca²⁺]_i through elevating extracellular Ca²⁺ or the addition of forskolin which increased [Ca²⁺]_i but desensitized the myofilaments to Ca²⁺.     

CHANGES IN PLATELET FREE Ca2+ CONCENTRATION AFTER CHRONIC DIGOXIN TREATMENT

Summary— Cell Na+ and Ca²⁺ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca²⁺ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca²⁺-countertransport system. Cytosolic free Ca²⁺ concentration is also controlled by the Na+–H+ exchange activity. To analyze the changes in cytosolic Ca²⁺ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca²⁺]_i was increased in platelets from 169±30 to 321±61 nmol/l ( n = 7, P <0.02) and unchanged in erythrocytes (121±6 and 104±7 nmol/l). This increase in platelet [Ca²⁺]_i was not accompanied by a change in serotonin content (5.43±0.67 vs 5.49±0.61 10⁻⁷ mol per 10¹¹ cells) and could not be reproduced by in vitro addition of 10⁻⁴ mol/l ouabain (198±33 vs 186±73 nmol/l). The enhanced [Ca²⁺]_i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca²⁺ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.     

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets

Summary— Na/Ca exchange was recently shown to regulate cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated ⁴⁵Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase ⁴⁵Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased ⁴⁵Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca²⁺ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.     

Insulin inhibits tissue factor expression in monocytes

Summary.  Objectives:  Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin. Methods and results:  LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1–100 nmol L⁻¹) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L⁻¹ insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 ± 2, 27 ± 3 and 52 ± 4% ( n  = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R – G_iα₂ complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G_i (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca²⁺, quenching of Ca²⁺ increases (BAPTA-AM) reduced and induction of Ca²⁺ entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca²⁺ mobilization but not with ATP-induced Ca²⁺ rises. Conclusions:  Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G_iα₂-mediated cAMP suppression, which attenuates Ca²⁺-mediated TF synthesis.     

Y27632, a Rho-activated kinase inhibitor, normalizes dysregulation in alpha1-adrenergic receptor-induced contraction of Lyon hypertensive rat artery smooth muscle

RhoA-activated kinase (ROK) is involved in the disorders of smooth muscle contraction found in hypertension model animals and patients. We examined whether the α1-adrenergic receptor agonist-induced ROK signal is perturbed in resistance small mesentery artery (SMA) of Lyon genetically hypertensive (LH) rats, using a ROK antagonist, Y27632. Smooth muscle strips of SMA and aorta were isolated from LH and Lyon normotensive (LN) rats. After Ca²⁺-depletion and pre-treatment with phenylephrine (PE), smooth muscle contraction was induced by serial additions of CaCl₂. In LH SMA Ca²⁺ permeated cells to a lesser extent as compared with LN SMA, while CaCl₂-induced contraction of LH SMA was greater than that of LN SMA, indicating a higher ratio of force to Ca²⁺ in LH SMA contraction (Ca²⁺ sensitization). No hyper-contraction was observed in LH aorta tissues. Treatment of LH SMA with Y27632 restored both Ca²⁺ permeability and Ca²⁺-force relationship to levels seen for LN SMA. In response to PE stimulation, phosphorylation of CPI-17, a phosphorylation-dependent myosin phosphatase inhibitor protein, and MYPT1 at Thr853, the inhibitory phosphorylation site of the myosin phosphatase regulatory subunit, was increased in LN SMA, but remained unchanged in LH SMA. These results suggest that the disorder in ROK-dependent Ca²⁺ permeability and Ca²⁺-force relationship is responsible for LH SMA hyper-contraction. Unlike other hypertensive models, the ROK-induced hyper-contractility of LH SMA is independent of MYPT1 and CPI-17 phosphorylation, which suggests that ROK-mediated inhibition of myosin phosphatase does not affect SMA hyper-contractility in LH SMA cells.     

Effects of some divalent cations on nitrergic relaxations in the mouse corpus cavernosum

Acute effects of some divalent cations (Cd²⁺, Ni²⁺, Co²⁺, Zn²⁺, Mn²⁺ and Sn²⁺) were investigated on neurogenic and endothelium-dependent relaxations in the isolated mouse corpus cavernosum. Neither neurogenic nor endothelium-dependent relaxation was affected by cations at the concentrations used (up to 100 μ M ), except Cd²⁺. Although Cd²⁺ (20 and 40 μ M ) did not cause any significant alteration in the acetylcholine- (ACh) or sodium nitroprusside- (SNP) induced relaxation, it inhibited electrical field stimulation- (EFS) produced relaxation significantly. Zn²⁺ and selenium could not reverse this inhibitory action. Cd²⁺ did block the EFS-evoked guanethidine-sensitive contraction in the presence of N^G-nitro- L -arginine. Elevation of external Ca²⁺ content significantly reduced the inhibitions due to Cd²⁺ on the EFS-induced relaxation and on the EFS-evoked guanethidine-sensitive contraction. In the Ca²⁺-omitted medium, EFS-induced relaxation disappeared, while acetylcholine-elicited relaxation resisted. Verapamil was ineffective on the relaxation produced by EFS or acetylcholine. However, it significantly diminished phenylephrine-induced contractions. These findings suggest that unlike other cations at the concentrations used in the present study, Cd²⁺ may have an effect on an external Ca²⁺-dependent mechanism at the neuronal level, and this effect may be responsible for its acute inhibitory action on the neurogenic relaxation in the mouse corpus cavernosum.     

The effect of respiratory and metabolic acid-base changes on ionized calcium concentration: in vivo and in vitro experiments in man and rat

Abstract. Correlation of ionized calcium concentration, [Ca²⁺] and blood pH has long been recognized. So far no distinction of the acid-base changes, i.e. respiratory changes or metabolic changes seemed necessary. The present study, with the use of a recently developed system for in vivo analysis of [Ca²⁺], and with in vitro experiments reinvestigates this question.
In a first series respiratory and metabolic changes were induced in rats. Changes of [Ca²⁺] (Δ[Ca²⁺]) and of plasma pH (ΔpH) were recorded continuously in vivo , plasma bicarbonate, [HCO^-₃] was measured in vitro . In a second series respiratory and metabolic changes were induced in sixteen volunteers and, separately, in vitro in plasma and modified Ringer solution, and the same parameters were determined.
In all experiments Δ[Ca²⁺] correlates negatively with ΔpH. However, the correlation in respiratory changes was significantly less as compared to that in metabolic changes. As expected, Δ[HCO^-₃] correlates positively with pH in metabolic and negatively in respiratory changes. We conclude from these experiments that in metabolic changes the effects of calciumalbumin interaction and calcium complexation with bicarbonate are additive, whereas both effects oppose each other in respiratory changes. This might explain the blunted effect of pH changes on [Ca²⁺] in respiratory changes.     

Blockers of NMDA-operated channels decrease glutamate and aspartate extracellular accumulation in striatum during forebrain ischaemia in rats

Summary— Brain microdialysis was used to study changes in the glutamate and aspartate extracellular concentrations in the striatum of conscious rats submitted to 30 minutes cerebral ischaemia, using the four-vessel occlusion model. Perfusion of the N-methyl-D-aspartate (NMDA) receptor channel blockers, dizocilpine (MK-801; 75 μM) and Mg²⁺ (2.5 mM), inhibited the ischaemia-induced accumulation of glutamate and aspartate. The AMPA/kainate receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamylbenzo (F) quinoxaline (NBQX; 15 μM and 450 μM) had no effect on glutamate and aspartate levels during ischaemia. On the other hand, omission of Ca²⁺ from the perfusing solution did not alter the increases in glutamate and aspartate induced by ischaemia. These results suggest that the glutamate and aspartate accumulation in four-vessel occlusion ischaemia is mediated by activation of NMDA receptors in a Ca²⁺ independent manner.     

Activated protein C attenuates coagulation-associated over-expression of fibrinolytic activity by suppressing the thrombin-dependent inactivation of PAI-1

Summary.  Several activated coagulation factors have been reported to enhance fibrinolysis by neutralizing plasminogen activator inhibitor type 1 (PAI-1) activity. We evaluated the physiological relevance of this mechanism using the euglobulin clot lysis time (ECLT) assay in the presence and absence of Ca²⁺, which is controlled by PAI-1 and mimics physiological thrombolysis. We found that the ECLT (18.5 ± 0.6 h) was shortened by Ca²⁺ (5 m m ) (6.6 ± 0.1 h). A significant difference was observed in thrombin generation by the presence of Ca²⁺ in the euglobulin fraction. Prothrombin was almost fully converted to thrombin within 15 min in the presence of Ca²⁺, whereas essentially no conversion was observed without Ca²⁺. The presence of activated protein C (aPC) suppressed thrombin generation, and attenuated the shortening of ECLT in a dose-dependent manner, an effect enhanced by phospholipid and protein S. In the absence of Ca²⁺, aPC did not prolong the ECLT. After addition of biotin-labeled recombinant PAI-1 to the euglobulin fraction, PAI-1 was cleaved to lower molecular weight forms only in the presence of Ca²⁺. This cleavage did not occur in the presence of aPC, suggesting that thrombin was the catalyst for PAI-1 cleavage. The cleavage and inactivation of PAI-1 by generated thrombin is proposed to be responsible for the shortening of ECLT by Ca²⁺ and for coagulation-associated over-expression of fibrinolysis. Under such conditions, aPC appeared to suppress thrombin generation and to normalize highly activated fibrinolysis.     

The critical role of magnesium ions in osteoclastmatrix interaction: implications for divalent cations in the study of osteoclastic adhesion molecules and bone resorption

Abstract. We have studied the adhesion and dissociation of isolated rat osteoclasts from plastic or bone matrix using time-lapse videomicroscopy and adhesion assays, and find that Mg²⁺ and not Ca²⁺ is essential for cell attachment and that Ca²⁺ and not Mg²⁺ is critical for dissociation.