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.     

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.     

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.     

Inhibited neutrophil functions in patients treated with nifedipine but not with verapamil or diltiazem

Neutrophil functions were studied in patients receiving calcium channel blockers: nifedipine, diltiazem or verapamil. Neutrophils from patients treated with nifedipine showed a significantly lower superoxide generation stimulated by phorbol myristate acetate (PMA) (50 ng mL⁻¹), opsonized zymosan (1 mg mL⁻¹) or formyl-methionyl-leucyl-phenylalanine (FMLP) (10⁻⁷  m ), whereas superoxide generation by neutrophils of patients receiving diltiazem or verapamil showed only a slight and insignificant reduction compared with controls. Similarly, chemotaxis towards 10⁻⁷  m FMLP and phagocytosis were significantly lower in patients receiving nifedipine compared with controls and were only slightly reduced in patients receiving diltiazem or verapamil. Nifedipine was the most efficient drug in inhibiting the rise in intracellular calcium ion concentration ([Ca²⁺]_i) when added in vitro and in neutrophils of patients receiving this drug, whereas verapamil had no significant effect. The correlation between the inhibitory effect of nifedipine on neutrophil function and the elevation of [Ca²⁺]_i suggests that nifedipine inhibits neutrophil functions through its effect on [Ca²⁺]_i. However, it is not the sole mechanism as superoxide generation induced by PMA, an agent that does not induce a rise in [Ca²⁺]_i, is also inhibited. The unique effect of nifedipine in reducing neutrophil functions in vivo suggests its clinical implications concerning response to acute ischaemic myocardial events.     

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.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications

Summary— Smooth muscle contraction is the basis of the physiological reactivity of several systems (vascular, respiratory, gastrointestinal, urogenital…). Hyperresponsiveness of smooth muscle may also contribute to a variety of problems such as arterial hypertension, asthma and spontaneous abortion. An increase in cytoplasmic calcium concentration ([Ca²⁺]_i) is the key event in excitation-contraction coupling in smooth muscle and the relationship linking the [Ca²⁺]_i value to the force of contraction represents the calcium sensitivity of the contractile apparatus (CaSCA). Recently, it has become evident that CaSCA can be modified upon the action of agonists or drugs as well as in some pathophysiological situations. Such modifications induce, at a fixed [Ca²⁺]_i value, either an increase (referred to as sensitization) or a decrease (desensitization) of the contraction force. The molecular mechanisms underlying this modulation are not yet fully elucidated. Nevertheless, recent studies have identified sites of regulation of the actomyosin interaction in smooth muscle. Sensitization primarily results from the inhibition of myosin light chain phosphatase (MLCP) by intracellular messengers such as arachidonic acid or protein kinase C. In addition, phosphorylation of thin filament-associated proteins, caldesmon and calponin, increases CaSCA. Activation of small (monomeric) G-proteins such as rho or ras is also involved. Desensitization occurs as a consequence of phosphorylation of myosin light chain kinase (MLCK) by the calcium-calmodulin activated protein kinase II, or stimulation of MLCP by cyclic GMP-activated protein kinase. In the present review, examples of physiological modulation of CaCSA as well as pharmacological and pathophysiological implications are illustrated for some smooth muscles.     

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⁺.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Activation of the respiratory burst in eosinophil leucocytes—a transduction sequence decoupled from cytosolic Ca2+ rise

Abstract. The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5 nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca²⁺]_i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca²⁺]_i, it restored the burst response to agonists in Ca²⁺-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca²⁺-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G_i-proteins, but that in the presence of PMA portions of the agonist response could be recovered.     

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.     

Flavonoid inhibition of platelet procoagulant activity and phosphoinositide synthesis

Summary.  Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection. Phosphatidylinositol 4,5-bisphosphate (PIP₂) was previously reported to play a direct role in phosphatidylserine (PS) exposure, as a Ca²⁺ target. Thrombin formation and platelet procoagulant activity are dependent on PS exposure. As flavonoids can inhibit phosphoinositide (PPI) kinases, we examined whether changes in PPI metabolism in flavonoid-treated platelets could be involved in their antiplatelet effects. Treatment with the flavonoids quercetin or catechin reduced PS exposure, thrombin formation, PIP₂ level and resynthesis after platelet activation with collagen, thrombin or calcium ionophore. Flavonoids also prevented [Ca²⁺]_i increase induced by collagen, but not by the ionophore. The ability of flavonoids to decrease PS exposure induced by ionophore treatment could result from the diminution of PIP₂ levels, whereas PS exposure induced by collagen could also be diminished by flavonoids' effects on calcium signaling dependent on PIP₂ hydrolysis. These data favor a role for PIP₂ in the antiplatelet effects of flavonoids.     

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.     

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.     

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.     

Serum β2-microglobulin at remission and relapse in patients with multiple myeloma

Abstract. Serum β₂-microglobulin (S-β₂m) was determined before treatment in fifty patients with multiple myeloma (MM), twenty-two of which had pure Bence Jones (BJ) myeloma. S-β₂m was related to clinical stage before, but not after, a correction of S-β₂m values for co-existent raised S-creatinine values (> 106 μmol 1^-1)- However, S-β₂m as well as corrected β₂m was a parameter of prognostic value. The median expected survival time was 14 months at S-β₂m values > 8·0 mg 1^-1 and 56 months at values<3·5 mg 1^-1. A response to treatment was associated with a decrease of S-β₂m and a stationary course with unchanged values, whereas a relapse or progressive disease was connected with an increase. In β₂m producers', i.e. patients with a clear initial high S-β₂m, serial determinations are of value for monitoring patients with MM. In particular, this is the case in BJ myelomas, as they lack a quantifiable serum M-component. With respect to β₂m no difference was found between BJ and other patients with MM.     

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.     

α1-adrenoceptor subtype(s) mediating noradrenaline-induced contractions of the guinea-pig aorta

Summary— The effect of α₁-adrenoceptor subtype selective antagonists, WB 4101, SZL-49 and chloroethylclonidine on noradrenaline-induced contractions of the guinea-pig aorta has been studied in an attempt to identify the α₁-adrenoceptor subtype(s) involved in the response. Noradrenaline and SDZ NVI 085 induced contractions of the aorta. Noradrenaline-induced contractions were competitively antagonised by WB 4101 (pA₂ = 8.92, slope = 1.05). The contractions were significantly reduced by SZL-49 but not by chloroethylclonidine, indicating an action on α_1A-adrenoceptor subtype. Noradrenaline-induced contractions of the aorta were not inhibited by nifedipine (10⁻⁶ M). The results are interpreted to suggest that α_1A-adrenoceptor subtype mediates noradrenaline-induced contractions of the guinea-pig aorta and that activation of α_1A-adrenoceptor subtype in the guinea-pig aorta is probably linked to intracellular Ca⁺⁺ release.