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.     

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.     

Mast cell activation involves plasma membrane potential- and thapsigargin-sensitive intracellular calcium pools

Summary— The regulation and role of the intracellular Ca²⁺ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca²⁺]i) was monitored in fura-2 loaded mast cells. In the presence of Ca²⁺ and K+, compound 48/80 induced a biphasic increase in [Ca²⁺]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn²⁺. DTPA, a cell-impermeant chelator of Mn²⁺, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca²⁺, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca²⁺ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca²⁺, showing that a Ca²⁺ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca²⁺ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane.     

Paradoxical inhibitory effect of cromakalim on 86Rb outflow from pancreatic islet cells

Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the "K+ channel openers." The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ 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.     

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.     

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.     

Similarities between the effects of pinacidil and diazoxide on ionic and secretory events in rat pancreatic islets

The present study aimed at comparing the effects of pinacidil, a putative K+ channel opener, and diazoxide on ionic and secretory events in rat pancreatic islets. Pinacidil and diazoxide provoked a dose-dependent increase in 86Rb outflow from pancreatic islets perifused in the presence of glucose. Both drugs inhibited the glucose- and tolbutamide-induced increase in 45Ca outflow and insulin release whereas failing to affect the ionic and secretory responses to K+ depolarization. Pinacidil and diazoxide, in contrast to quinine, failed to affect 86Rb outflow from pancreatic islets stimulated by the Ca++-ionophore A23187. Pinacidil as well as diazoxide abolished the glucose-induced increase in [Ca++]i but did not modify the rise in [Ca++]i provoked by KCl. Lastly, both drugs were shown to stimulate an ouabain-resistant modality of 86Rb inflow into the islet cells. The close similarities between the ionic and secretory events mediated by pinacidil and diazoxide suggest that pinacidil could interfere with the same target site as diazoxide; namely the beta-cell ATP-sensitive K+ channel.     

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.     

Novel molecules in calcium signaling in platelets

Summary.  A rise in the intracellular calcium (Ca²⁺) concentration is a major component of the signaling mechanisms regulating platelet function in thrombosis and hemostasis. Previous studies, however, failed to identify many key molecules regulating Ca²⁺ signaling in platelets. Here, we review recent findings, which identified CalDAG-GEFI as a critical Ca²⁺ sensor that links increases in intracellular Ca²⁺ to integrin activation, TxA₂ formation, and granule release in stimulated platelets. Furthermore, we summarize work that lead to the discovery of STIM1 and Orai1 as key regulators of store-operated calcium entry (SOCE) in platelets. A short discussion on the usefulness of each molecule as a potential new target for antiplatelet therapy is included.     

Effects of tacrine on insulin secretion and 86Rb+ and 45Ca++ efflux from rat pancreatic islets.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine), a drug that has attained interest because of its ability to alleviate symptoms in Alzheimer's type of dementia, was found to stimulate insulin secretion from isolated rat pancreatic islets. The insulinotropic effect of the drug was observed at 8.3 mM but not at 3.3 mM glucose and was dependent on extracellular Ca++. From perifused 86Rb(+)-prelabeled islets, tacrine inhibited the fractional efflux of 86Rb+ at 3.3 mM glucose, but stimulated 86Rb+ efflux at 8.3 mM glucose. These effects persisted in the absence of extracellular Ca++. Tacrine also stimulated 45Ca++ efflux from perifused 45Ca(++)-prelabeled islets at 8.3 mM but had no effect on 45Ca++ efflux at 3.3 mM glucose or in the absence of extracellular Ca++. It is concluded that tacrine potentiates glucose-stimulated insulin secretion by a mechanism that is dependent on extracellular Ca++ and involves an increased Ca++ influx. The increased Ca++ influx is either secondary to a decreased K+ permeability induced by an inhibition of ATP-dependent K+ channels and/or due to a direct effect of tacrine on glucose-activated Ca++ channels.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

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⁺.     

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.     

Insulinotropic effect of new glibenclamide isosteres

The aim of the present study was to characterize the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano- N"- cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene), two newly synthesized isosteres of glibenclamide, on ionic and secretory events in rat pancreatic islet cells. Both compounds inhibited 86Rb (42K substitute) outflow from rat pancreatic islets perifused throughout at low (2.8 mM) D-glucose concentration. In excised inside-out membrane patches, BM 208 and BM 225 reduced the frequency of KATP+ channel openings. The inhibition of 86Rb outflow induced by BM 208 and BM 225 coincided with an increase in 45Ca outflow. The latter phenomenon was abolished in islets exposed to Ca2+-free media. Both isosteres of glibenclamide increased the [Ca2+]i in single pancreatic islet cells. This effect was counteracted by verapamil, a Ca2+ entry blocker. In islets exposed to 2.8 mM glucose and extracellular Ca2+, BM 208 and BM 225 stimulated insulin output. The secretory capacity of BM 225 was more marked than that of BM 208, but the time courses of the cationic and secretory responses exhibited obvious dissociations. These data suggest that the secretory capacity of BM 208 and BM 225 results, at least in part, from the inhibition of ATP-sensitive K+ channels with subsequent increase in Ca2+ inflow. The dissociation between cationic and secretory variables further suggests that the modifications in Ca2+ handling are not solely attributable to a primary inhibition of the ATP-sensitive K+ channels.     

Alzheimer amyloid β-peptides exhibit ionophore-like properties in human erythrocytes

Abstract. There is growing evidence that the amyloid β-peptide (β₁_₄₀) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta₁_₄₀ has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether β₁-₄₀ (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2h and 4h at 37°C together with 6μmolL^-1 of β₁-₄₀ or of fragments β₁_₂₈and β₂₅-₃₅, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05μmolL^-1). Moreover, β₁_₄₀ and its two fragments induced a significant alteration of ⁴⁵Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of β₁_₄₀ and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca²⁺-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease.     

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.     

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 effect of LU-49938 (nexopamil) on the activation by serotonin of mesangial cells

Summary— Intraglomerular platelet activation may release vasoactive agents such as serotonin (5-hydroxytryptamine, 5-HT) that may affect local hemodynamics and promote mesangial proliferation, eventually leading to glomerular sclerosis. The main purpose of this study is to analyze whether nexopamil, a verapamil derivative, with the property of blocking simultaneously calcium channels and 5-HT₂ receptors, could modify the contractile and mitogenic effects of serotonin on cultured rat mesangial cells. Serotonin caused a concentration-dependent increase in [³H]thymidine incorporation into DNA, and mesangial cell proliferation. The effects of 5-HT on thymidine uptake and cell proliferation were blocked by the selective 5-HT₂ receptor blocker ketanserin 10⁻⁵ M. Nexopamil abolished in a concentration-dependent way the serotonin-induced [³H]thymidine incorporation into DNA, and the serotonin-induced increase in number of cells. Using 5-HT 10⁻⁴ or 10⁻⁵ M, nexopamil had significant effects at concentration above 10⁻⁷ M. Serotonin induced a concentration- and time-dependent reduction of planar cell surface area. This effect was also completely blocked by ketanserin. Nexopamil partially blocked the serotonin-induced mesangial cell contraction, in a dose-dependent manner. All these data suggest that nexopamil inhibits both 5-HT-induced mesangial cell contraction and proliferation by blocking 5-HT₂ receptors and the voltage-operated Ca²⁺ channels.