Identification and characterization of α1- and β2-adrenergic receptors in human liver

Adrenergic receptors were identified in healthy human hepatic tissue from thirty-nine subjects undergoing elective abdominal surgery by using the specific alpha 1-antagonist [3H]-prazosin and the beta adrenergic antagonist [3H]-dihydroalprenolol ([3H]-DHA). [3H]-prazosin binding to plasma membranes was rapid, of high affinity, saturable and stereospecific with a maximal binding capacity (Bmax) of 74.1 +/- 5.5 fmol mg-1 of protein. The displacement curve for (-)-norepinephrine was better explained by a one-site binding and after addition of GTP 0.1 mM the curve was not right-shifted, suggesting the majority of alpha receptors in healthy human liver are of the alpha 1 subtype and not linked to a GTP-binding protein. [3H]-DHA binding to liver plasma membranes was also rapid, of high affinity, saturable and stereospecific with a Bmax 96.5 +/- 10.3 fmol mg-1 of protein of receptors. Computer aided analysis of the displacement curve of ICI 118,551, a subtype selective beta 2-antagonist (IC50 = 62 +/- 2 nM), indicated a one-site binding, thus, showing that beta adrenergic receptors are of the beta 2 subtype. The displacement curve of [3H]-DHA for (-)-isoproterenol was right shifted by GTP indicating that beta 2 adrenergic receptors are linked to a GTP-binding protein in human liver. These results indicate that alpha 1- and beta 2-receptors co-exist in human liver but only beta 2-receptors are linked to a GTP-binding protein.     

α2-Antiplasmin is involved in the production of transforming growth factor β1 and fibrosis

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.     

Platelet α2- and leucocyte β2-adrenoceptors in phaeochromocytoma: effect of tumour removal

Platelet alpha 2- and leucocyte beta 2-adrenoceptors (as well as noradrenaline and adrenaline plasma levels) were studied in five patients with confirmed phaeochromocytoma using tritiated yohimbine and iodated cyanopindolol, respectively, before and after tumour removal. Patients with phaeochromocytoma had a lower leucocyte iodated cyanopindolol binding than control subjects, but no change in platelet tritiated yohimbine binding. Plasma catecholamine levels were higher than controls. Tumour removal induced a return to normal values of leucocyte iodated cyanopindolol binding and a decrease in plasma catecholamine levels. These results underline the potential interest of leucocyte beta-adrenoceptor quantification in the diagnosis and the follow-up of phaeochromocytoma. Pharmacologically, they show that down-regulation occurs in vivo for leucocyte beta 2-adrenoceptors but not for platelet alpha 2-adrenoceptors.     

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.     

NORADRENALINE RELEASE IN HUMAN CORPUS CAVERNOSUM AND ITS MODULATION VIA PRESYNAPTIC α2-ADRENOCEPTORS

Strips of human corpus cavernosum were incubated with 3H-noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. The electrically (0.66 Hz) evoked tritium overflow was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid. The alpha 2-adrenoceptor agonist B-HT 920 (6-allyl-2-amino-5, 6, 7, 8-4H-thiazolo-5,4-d-azepine), inhibited the evoked overflow, whereas the alpha 1-adrenoceptor agonist, methoxamine, was ineffective. The alpha 2-adrenoceptor antagonist, rauwolscine, facilitated the evoked tritium overflow. It is concluded that the stimulation-evoked release of noradrenaline from the sympathetic nerve fibres of the human corpus cavernosum is modulated via presynaptic alpha 2-adrenoceptors.     

MMP-2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary. Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active α_IIbβ₃. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin α_IIbβ₃ in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the α_IIbβ₃‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin α_IIbβ₃ was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active α_IIbβ₃, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin α_IIbβ₃. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to α_IIbβ₃.     

α1 and α2-adrenergic control of large and small coronary arteries during exercise in conscious dogs under β-blockade

Summary— The aim of this study was to determine the relative roles of α₁-and α₂-adrenoceptors at the level of large epicardial and small resistance coronary arteries when sympathetic tone is increased by exercise in conscious dogs. The responses of left circumflex coronary artery diameter and blood flow were investigated at rest and during graded treadmill exercise (5, 10 and 12 km/h) in six chronically instrumented dogs during control conditions (saline) and after administration of propranolol (1 mg/kg) either alone or in combination with either prazosin (50 μg/kg), or idazoxan (300 μg/kg), or the association of prazosin + idazoxan (same doses). In control conditions, graded treadmill exercise resulted in a progressive increase in coronary artery diameter (+ 3.8 ± 0.6% from 3479 ± 80 μm) and in a decrease in coronary vascular resistance (- 46.0 ± 4.5% from 8.49 ± 1.51 mmHg/cm/s). Propranolol significantly constricted large (- 4.4 ± 0.6% from 3486 ± 87 μm) and limited dilation of small coronary arteries during exercise. These coronary effects of propranolol remained unchanged following additional α₂-adrenoceptor blockade by idazoxan but were abolished following α₁-adrenoceptor blockade by prazosin, given either alone or combined with idazoxan. Thus, α₁- but not α₂-adrenoceptors are responsible for propranolol-induced constriction of large coronary arteries and limitation of small coronary arteries dilation during exercise in conscious dogs.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

Changes in plasma glucose and lactate evoked by α and β2-adrenoceptor stimulation in conscious fasted rabbits

In conscious fasted rabbits, the iv infusion of salbutamol (3 micrograms/kg per min) and clonidine (2 micrograms/kg per min) induced a blood glucose increase amenable to blockade, respectively by ICI 118551 (1 micrograms/kg per min) and idazoxan (20 micrograms/kg per min). Amidephrine (10 micrograms/kg per min) and salbutamol mediated an increase in plasma lactate which was attenuated by prazosin (50 micrograms/kg, sc) and ICI 118551 respectively. Clonidine did not alter basal plasma lactate. The iv infusion of adrenaline (0.3 micrograms/kg per min) evoked an increase in plasma lactate more sensitive to blockade by ICI 118551 than by prazosin. ICI 118551 also shortened the hyperglycaemic response to adrenaline, 3-Mercaptopicolinic acid (25 mg/kg) reduced salbutamol- and adrenaline-mediated hyperglycaemia and increased at the same time the lactate/glucose ratio. Our data show that plasma lactate levels may be regulated by alpha 1- and beta 2-excitatory adrenoceptor stimulation. However, only the increase in blood lactate derived from beta 2-adrenergic stimulation seems to contribute to the overall catecholamine-mediated hyperglycaemia.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

Platelet integrin αIIbβ3: activation mechanisms

Integrin alpha(IIb)beta(3) plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of alpha(IIb)beta(3) depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of alpha(IIb)beta(3) and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of alpha(IIb) and beta(3) cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of alpha(IIb)beta(3) activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation.     

α2-Macroglobulin reduces paracrine- and autocrine-stimulated matrix synthesis of cultured rat hepatic stellate cells

BACKGROUND: Transforming growth factor beta1 (TGF-beta1) is considered to represent a major fibrogenic mediator in the liver. The aim of the present study was to investigate whether alpha2-macroglobulin (alpha2M) might reduce paracrine- and autocrine-stimulated matrix synthesis of cultured rat hepatic stellate cells (HSCs) by scavenging TGF-beta. METHODS AND RESULTS: Using native agarose electrophoresis, we demonstrated that alpha2M binds [125I]-TGF-beta1 within minutes. Preincubation of transiently acidified supernatants of cultured Kupffer cells, secondary cultured (activated) HSC and platelet lysate with, respectively, 500 and 2000 microg mL-1 alpha2M significantly reduced the concentration of active TGF-beta1 in these media. As a consequence of TGF-beta scavenging by alpha2M, paracrine-stimulated proteoglycan synthesis of primary cultured HSCs was reduced significantly. Furthermore, addition of 200 microg mL-1 alpha2M to passaged (activated) HSCs resulted in (a) a reduction in autocrine-stimulated extracellular matrix synthesis (proteoglycan -52%, fibronectin -55%) and (b) increased cell proliferation. A similar reduction in matrix synthesis was observed after the addition of 5 micromol L-1 TGF-beta1 antisense oligonucleotide to activated HSCs. CONCLUSION: We conclude that alpha2M reduces paracrine-and autocrine-stimulated extracellular matrix synthesis of cultured HSCs by scavenging TGF-beta. These mechanisms might restrict liver fibrogenesis.     

Peripheral α2-adrenoceptor-mediated sympathoinhibitory effects of mivazerol

Summary— Mivazerol is a new compound that could potentially reduce perioperative cardiovascular morbidity and mortality in patients with or at risk of coronary disease and submitted to surgery. This action of mivazerol depends on a well documented centrally mediated reduction in sympathetic nerve activity, but a direct peripheral decrease in sympathetic neurotransmitter release induced by activation of prejunctional α₂-adrenoceptors located on sympathetic nerve endings could also contribute. To investigate this issue, the effects of mivazerol on the pressor, systemic and regional hemodynamic (pulsed Doppler technique) as well as on the cardiac responses to electrical stimulation of the spinal cord (SCS) were measured in pithed rats in the absence and in the presence of mivazerol. Mivazerol exerted strong sympathoinhibitory effects: SCS-induced increases in blood pressure, total peripheral resistance and heart rate were dose-dependently reduced by mivazerol, but among the regional vascular beds investigated, only the hindlimb vasoconstrictor responses were significantly drug-affected. All these sympathoinhibitory effects of mivazerol were abolished by prior yohimbine administration. Simultaneously, mivazerol did not induce any postjunctional adrenoceptor blockade as it did not affect noradrenaline cardiac and hemodynamic effects. On the contrary, through postjunctional α₂-adrenoceptor stimulation, mivazerol, in this pithed preparation, dose-dependently increased blood pressure, total peripheral and hindlimb vascular resistances, but heart rate was not affected. We conclude that, in the pithed rat, mivazerol exerts strong peripheral sympathoinhibitory effects. The mechanism involved is prejunctional α₂-adrenoceptor activation as i) mivazerol does not display any postsynaptic α-adrenoceptor blocking effect — it even behaves as a postsynaptic α₂-adrenoceptor agonist — and ii) yohimbine abolishes mivazerol's sympathoinhibitory effects. Thus, direct peripheral together with central mechanisms contribute to mivazerol's sympathoinhibitory effects and ultimately to its cardioprotective action.     

Differential activation of β1-, β2- and β3-adrenoceptors by catecholamines in white and brown adipocytes

Summary— This review summarizes the experiments performed by various groups to determine how the activation of the different β-adrenoceptors (β-ARs) is ordinated when they are present in the same fat cell and involved in the same biological event. When expressed after the transfection of their genes in Chinese hamster ovary cell (CHO cells), β₁- and β₂-ARs present a higher affinity for catecholamines than β₃-ARs. In vitro, the lipolytic effect induced by low concentrations of catecholamines in dog and rat white fat cells is due to the selective activation of β₁- and/or β₂-ARs. Higher concentrations only are able to activate β₃-ARs. Similar results have been obtained in rat brown adipocytes. On the other hand, the lipolytic effect of catecholamines in human and primate adipocytes does not involve a β₃-AR component whatever the concentration used. In vivo experiments in the dog have also shown that lipomobilization induced by low doses of isoprenaline only involved β₁- and β₂-AR activation, this effect being blocked by β₁-/ β₂-antagonist pretreatment. However, in the same blockade conditions, perfusion of a 10-fold higher dose of isoprenaline revealed a β₃-AR contribution in the lipomobilizing effect. These data showed that brown and white adipocyte β₃-ARs possess a lower affinity for catecholamines than β₁- and β₂-ARs and are only recruited by high concentrations of the amines. Thus, even if the β₃-AR plays an indisputable role in the white and brown adipose tissue of some species, its physiological status and its expression are still subject to debate in human white fat cells.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.     

Increased α2- but similar β-adrenergic receptor activities in subcutaneous gluteal adipocytes from females compared with males

alpha 2 and beta-adrenergic binding and action were studied in subcutaneous adipocytes from the gluteal region in females and males. The beta-selective antagonist [3H]dihydroalprenolol and the alpha 2-selective antagonist [3H]yohimbine were used to identify the beta- and the alpha 2-receptor, respectively. The biological effects mediated by these receptors were determined by measuring the glycerol release induced by isoproterenol (beta-receptor agonist) and by clonidine (alpha 2-receptor agonist). The study consisted of health volunteers (eighteen females and fifteen males). Compared to men the alpha 2-receptor binding was increased by 73% (P less than 0.01) in adipocytes from females. From Scatchard analysis it was determined that the increased binding was due to an increased receptor number (438 v. 262 fmol mg-1 protein, P less than 0.001) with unaltered Kd (1.18 v. 1.35 nmol l-1, P greater than 0.05). This increased alpha 2-receptor binding in female and adipocytes was associated with a significantly increased sensitivity (P less than 0.05) and maximal antilipolytic effect of clonidine (P less than 0.05). The beta-receptor binding was similar in adipocytes from females and males. However, isoproterenol was significantly more lipolytic in female adipocytes (P less than 0.01). Since the combination of theophylline and adenosine deaminase also was significantly more lipolytic in female adipocytes the enhanced effect of isoproterenol then seemed to be due to mechanisms not directly related to the hormone-beta-receptor binding. Finally, the mixed beta- and alpha 2-receptor agonist, adrenaline showed biphasic effects on lipolysis, with stimulatory effect at low concentrations (less than 500 nmol l-1) and pronounced inhibitory effect at higher concentrations (greater than 1 mumol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

α-Methylnoradrenaline-induced contractions in rat aorta are mediated via α1D-adrenoceptors

Summary— The subtype(s) of α-adrenoceptor-mediating contractions to α-methynoradrenaline in the rat aorta has been investigated by using α-adrenoceptor-selective competitive antagonists and the α₁-adrenoceptor selective agonist, phenylephrine, for comparison. α-Methylnoradrenaline and phenylephrine elicited concentration-dependent contractions in the endothelium-denuded and endothelium-intact aortic rings with similar potencies and maximal effects. α-Methylnoradrenaline- and phenylephrine-induced contractions in endothelium-denuded aortic rings were competitively antagonized by prazosin (pA₂ = 9.38 and 9.13; respectively) and rauwolscine (pA₂ = 7.19 and 6.60, respectively). This confirms that there is an α₁- and a non α₂-adrenoceptor response to α-methylnoradrenaline in the rat aorta. The subtype selective α_1D-adrenoceptor antagonist, BMY 7378, was found to antagonize contractions to α-methylnoradrenaline and phenylephrine competitively in endothelium-denuded and endothelium-intact aortic rings. The pA₂ values of BMY 7378 against α-methylnoradrenaline (8.39 and 8.41; endothelium-intact and endothelium-denuded, respectively) and phenylephrine (8.64 and 8.76; endothelium-intact and endothelium-denuded, respectively), are consistent with its published functional potency and clonal α_1d-adrenoceptor binding affinity. In addition, contractions to α-methylnoradrenaline and phenylephrine in endothelium-denuded aortic rings, were potently inhibited by WB 4101 with pA₂ values of 9.75 and 9.25, respectively. The high pA₂ values for WB 4101 indicate that the α_1B-adrenoceptor subtype does not seem to participate in α-methylnoradrenaline (and phenylephrine) induced contractions in this artery. These results suggest that the α_1D-subtype plays a determining role in rat aorta contractions induced by α-methylnoradrenaline.     

Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.     

Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin αIIbβ3

BACKGROUND: We previously demonstrated that Dok2 is rapidly phosphorylated on tyrosine residues in platelets in response to thrombin, the immunoreceptor tyrosine-based activation motif-coupled collagen receptor glycoprotein (GP) VI, and by integrin alphaIIbbeta3. OBJECTIVES AND METHODS: In this study we further delineate the regulation of phosphorylation of Dok2 and compare this to the related adapter Dok1. RESULTS: We demonstrate expression of Dok1 in platelets and the unexpected observation that the adapter protein undergoes tyrosine phosphorylation in response to thrombin but not to GPVI or integrin alphaIIbbeta3. Furthermore, Dok1 phosphorylation is transient, peaking at 30 s and returning to basal by 5 min, whereas Dok2 phosphorylation is delayed but sustained. Dok2 phosphorylation, but not that of Dok1, is inhibited by Src kinase inhibitors and by chelation of intracellular calcium. Further, phosphorylation of Dok2 by thrombin and integrin alphaIIbbeta3 in mouse platelets is independent of Syk and phospholipase Cgamma2. Additionally, Dok2 coimmunoprecipitates with integrin alphaIIbbeta3 downstream of Src kinases. CONCLUSIONS: These results demonstrate differential modes of regulation of Dok1 and Dok2 in platelets. Further, they raise the interesting possibility that Dok2 plays an important role in integrin outside-in signaling through a physical and functional interaction with integrin alphaIIbbeta3.