Effect of castration on the concentration of adenosine 3′, 5′-monophosphate in the rat pineal organ

Summary The concentration of adenosine 3, 5-monophosphate (cAMP) was investigated in the rat pineal organ after bilateral orchidectomy. Orchidectomy caused a decrease in pineal cAMP concentration.This paper has been supported by a grant of the Polish Academy of Sciences, No. 10.4.2.01.5.6.     

Effects of adenosine 3′, 5′-cyclic monophosphate on thermoregulation in both rats and rabbits

Summary The effects of intraventricular administration of dibutyryl adenosine 3, 5-cyclic monophosphate (db cyclic AMP) on the thermoregulatory responses of unanesthetized rats and rabbits to different ambient temperatures (T_a) were assessed. Administration of db cyclic AMP (10–60 mM) produced dose-dependent hypothermia in both rats and rabbits at T_a 2–22 °C. The hypothermia in response to db cyclic AMP was due to decreased metabolic heat production and cutaneous vasodilatation. There was no change in respiratory evaporative heat loss. In contrast, in the heat (30–32 °C), db cyclic AMP administration produced dose-dependent hyperthermia in these animals. The hyperthermia was due to increased metabolism (due to muscular shivering) and decreased heat losses. The reduction in heat losses was shown by a decrease in both cutaneous circulation and respiratory evaporative heat loss. The data demonstrate that the thermoregulatory responses induced by central administration of db cyclic AMP are T_a-dependent.     

Cyclic adenosine 3′,5′monophosphate in cerebrospinal fluid of multiple sclerosis patients

Summary Cyclic adenosine 3,5monophosphate (cAMP) was assayed in CSF and plasma obtained from patients with multiple sclerosis. Decreased CSF cAMP levels were found in more than half of the patients while plasma cAMP was normal. The decrease is correlated significantly with the disability of the patient and with the progression of the disease. A low CSF cAMP level can be considered as prognostically unfavorable, particularly in the early stage of the disease. There was no correlation between the cAMP levels and the duration of the disease or with bouts and remissions. ACTH therapy did not normalize the decreased values. Obviously the decrease of CSF cAMP is related to the demyelination and not to the intensity of the pathological immunoreactions.

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Zusammenfassung Es wurde das cyclische Adenosin-3,5monophosphat im Liquor von Patienten mit multipler Sklerose untersucht. Bei einem Teil der Patienten wurden auch die Vergleichswerte im Blutplasma bestimmt. Es zeigte sich bei mehr als der Hälfte der Patienten eine Verminderung der cAMP-Konzentration im Liquor bei normalem Plasmaspiegel. Diese cAMP-Verminderung erwies sich als signifikant abhängig von dem Schweregrad der Erkrankung bzw. der Erkrankungsprogredienz und ist besonders in frühen Erkrankungsfällen als prognostisch ungünstiges Zeichen anzusehen. Es fand sich kein Zusammenhang mit dem Krankheitsstadium, d.h. Schub bzw. Intervall, und mit der Erkrankungsdauer. Eine ACTH-Behandlung vermochte diese Verminderung der Werte nicht auszugleichen. Es wird die Wertigkeit dieser Befunde diskutiert.

     

Effects of chronic antidepressant treatment on α1- and α2-adrenoceptors in the rat anococcygeus muscle

Summary The effects of a single administration (48 hours) and of chronic (14 days) treatment with tricyclic (desipramine, nortryptiline) and nontricyclic (mianserin, nomifensine) antidepressant drugs on responses of the isolated anococcygeus muscle to the ₂-adrenoceptor agonist xylazine (inhibition of contraction to field stimulation at 1 Hz) and to the ₁-adrenoceptor agonist phenylephrine (contraction of the muscle) have been studied.Of the drugs used only desipramine and nortryptiline administered chronically reduced the responsiveness of the anococcygeus muscle to phenylephrine suggesting a desensitization of postsynaptic ₁-adrenoceptors. Long-term but not acute administration of antidepressants resulted in significant decrease in sensitivity of presynaptic ₂-adrenoceptors to xylazine. These results show that the adaptative changes of -adrenoceptors in the rat anococcygeus muscle following long-term administration may depend on the efficiency to inhibit the neuronal uptake and the ability to antagonize ₁-adrenoceptors.     

Nicotinic acetylcholine receptors containing subunits α3 and α5 in rat nociceptive dorsal root ganglion neurons

Nociceptive primary afferent neurons carry nicotinic acetylcholine receptors (nAChRs). Using RTPCR, mRNAs for all alpha-subunits have been identified in rat dorsal root ganglia (DRG) (Genzen et al., 2001; Lips et al., 2002), but the responses of nociceptive neurons to nicotine are not uniform and the cellular distribution of nAChRs within DRG, in general, and among functionally different subtypes of primary afferent neurons, in particular, are only partially resolved (Rau et al., 2005). These diverse actions might suggest the presence of various nAChR isoforms that are operative under different conditions. The present study was aimed to extend previous studies on nAChRs that contain subunits alpha4, alpha7, and alpha10 in providing data for alpha3- and alpha5-subunit-containing nAChRs (Haberberger et al., 2004; Papadopolou et al., 2004). To this end, calcium-imaging and double-labeling immunofluorescence with nAChR alpha-subunit-specific antibodies, in combination with markers for nociceptive neurons (TRPV1, I-B4), were applied.     

S-100 protein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human brain tumors

Summary Biopsy specimens from 23 human brain tumors have been analyzed for the nervous system specific protein S-100 and the membrane-associated enzyme 2,3-cyclic nucleotide 3-phosphohydrolase (CpNase). Biopsy specimens from an additional seven brain tumors were tested for either S-100 or CpNase alone.All astrocytomas and glioblastomas tested were found to contain S-100 and CpNase although there does not appear to be a strong correlation between the levels of these two markers in the 14 such tumors assayed for both. S-100 levels varied over a 19-fold range while CpNase varied over a 835-fold range. Postoperative survival in the astrocytoma and glioblastoma patients showed only a weak correlation with either tumor CpNase or S-100 levels.Two acoustic neurinomas, two oligodendrogliomas, one mixed glioma, and one choroid plexus papilloma were also assayed and found to have detectable levels of both S-100 and CpNase with the acoustic neurinomas and the mixed glioma having relatively high levels of each marker. All six meningiomas tested had low levels of CpNase. S-100 assays in three benign meningiomas were negative, while low levels of this protein were found in the one malignant meningioma tested.Tissue cultures were grown out from biopsy specimens of additional human brain tumors and tested at confluency for S-100. Of 15 astrocytoma and glioblastoma cultures tested, three had easily detectable amounts of S-100, two appeared to contain trace levels and ten were negative. The two acoustic neurinoma cultures tested were positive for S-100 while all three oligodendroglioma cultures were negative.     

Involvement of postsynaptic α1- and α2-adrenoceptors in the flexor reflex activity in the spinal Rats

Summary Clonidine (0.2 mg/kg i.v.) and St 587 (1 mg/kg i.v.), selective agonsists of ₂- and ₁-adrenoceptors, respectively, increased the flexor reflex amplitude in the spinal rat. Yohimbine and rauwolscine, ₂-adrenoceptor antagonists, inhibited dose-dependently the effect of clonidine but not of St 587. However, prazosin and clozapine, ₁-adrenoceptor antagonists, diminished the action of both agonists in a dose-dependent manner. After central chemosympathectomy caused by 6-OH-DA or DSP-4 the response to clonidine did not differ from that in the sham-operated animals. In 6-OH-DA treated rats yohimbine (1 mg/kg i.v.) antagonized the effect of clonidine to the same extent as it did in unlesioned animals. It is concluded that the results are functional evidence that ₁- and ₂-adrenoceptors are present in the rat spinal cord, and stimulation of each receptor increases flexor reflex activity.     

The effect of chronic administration of amitriptyline on the effects of subsequent electroconvulsive treatment on responsiveness of α1- and β-adrenoceptors in the rat cortical slices

Summary Both antidepressant drugs and repeated electroconvulsive shock (ECS) produce adaptive changes in cerebral neurotransmitter systems. As in the clinical practice ECS is used almost always after therapeutical failure of pharmacotherapy, we investigated presently how chronic administration of an antidepressant amitriptyline affects the action of subsequent multiple ECS in rats. Amitriptyline differed from ECS and from other classical antidepressant in producing no -downregulation and potentiating the inhibitory effect of protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), on responses of ₁-adrenoceptor system to nor adrenaline. The action of ECS on ₁-adrenoceptor system remained essentially unaffected by previous amitriptyline administration. Its downregulatory effect on responses of -adrenoceptor system to nor adrenaline, and particularly to isoproterenol, were attenuated by previous drug treatment. The present results suggest that previous chronic administration of antidepressant drugs may alter the effect of subsequent ECS.     

Postnatal changes of nicotinic acetylcholine receptor 2, α3, α4, α7 and β2 subunits genes expression in rat brain

Postnatal changes of nicotinic acetylcholine receptor (nAChR) α2, α3, α4, α7 and β2 subunits mRNAs were investigated in rat brain using ribonuclease protection assay. Multiple developmental patterns were observed: (1) transient expression during the first few postnatal weeks; α2 in the hippocampus and brain stem, α3 in the striatum, cerebellum and cortex, α4 in the hippocampus, striatum and cerebellum, α7 in the cerebellum and β2 in the striatum. (2) Constant expression across development; α2 and α3 in the thalamus, α4 in the cortex, thalamus and brain stem, α7 in the thalamus and brain stem and β2 in all brain regions except striatum. (3) Non-detection across development; α2 in the cortex, striatum and cerebellum. (4) Increase with age; α7 in the cortex and hippocampus. (5) Bell-shaped development; α7 in the striatum. Postnatal changes of nAChR isoforms in different brain regions of rat were investigated by receptor binding assays. The developmental patterns of [³H]epibatidine and (−)-[³H]nicotine binding sites were similar to each other in each brain region, but different from that of [³H]α-bungarotoxin binding sites. No obvious correlation was observed between the developmental patterns of [³H]α-bungarotoxin, [³H]epibatidine and (−)-[³H]nicotine binding sites and corresponding subunits mRNAs. These results indicate that multiple mechanisms are involved in changes of gene expression of nAChRs subunits in the brain of developing rats.     

Neuronal and glial accumulation of α- and β-synucleins in human lipidoses

A number of the lysosomal storage diseases that have now been characterized are associated with intra-lysosomal accumulation of lipids, caused by defective lysosomal enzymes. We have previously reported neuronal accumulation of both α- and β-synucleins in brain tissue of a GM2 gangliosidosis mouse model. Although α-synuclein has been implicated in several neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, its functions remain largely unclear. In our present study, we have examined a cohort of human lipidosis cases, including Sandhoff disease, Tay–Sachs disease, metachromatic leukodystrophy, β-galactosialidosis and adrenoleukodystrophy, for the expression of α- and β-synucleins and the associated lipid storage levels. The accumulation of α-synuclein was found in brain tissue in not only cases of lysosomal storage diseases, but also in instances of adrenoleukodystrophy, which is a peroxisomal disease. α-synuclein was detected in both neurons and glial cells of patients with these two disorders, although its distribution was found to be disease-dependent. In addition, α-synuclein-positive neurons were also found to be NeuN-positive, whereas NeuN-negative neurons did not show any accumulation of this protein. By comparison, the accumulation of β-synuclein was detectable only in the pons of Sandhoff disease cases. This differential accumulation of α- and β-synucleins in human lipidoses may be related to functional differences between these two proteins. In addition, the accumulation of α-synuclein may also be a condition that is common to lysosomal storage diseases and adrenoleukodystrophies that show an enhanced expression of this protein upon the elevation of stored lipids.     

Interaction analysis between 5-HTTLPR and TNFA −238/−308 polymorphisms in schizophrenia

Summary. This study investigated the potential interaction between the polymorphisms of serotonin transporter gene (SLC6A4, a 44 base pair insertion/deletion in the promoter region, 5-HTTLPR) and tumor necrosis factor-α gene (TNFA; −238G/A and −308G/A polymorphisms) on the development of schizophrenia, as well as the interaction of the three polymorphisms in relation to symptomatology, family history, onset age and antipsychotic treatment response. Genomic DNA analyses with polymerase chain reaction (PCR) was used for the genotyping. One hundred and fifty-two (152) patients with schizophrenia and 152 normal controls participated in the study. Any associations between the individual polymorphism and schizophrenia were not found. However, marginal association between subjects with both TNFA −238 A allele (genotype AA plus AG) and 5-HTTLPR s allele (ss plus sl) and presence of family history was found (p = 0.023; p = 0.026). The subjects with TNFA −308 AG genotype showed higher change in PANSS total score (p = 0.028). No significant interaction effect between 5-HTTLPR and TNFA −238/−308 polymorphisms either on the development of schizophrenia or on antipsychotics treatment response and psychopathology was found, although a significant interaction effect for subjects carrying TNFA −238 AG and −308 AA genotypes on a positive family history was observed (p = 0.017). These results suggest that the interaction effects between 5-HTTLPR and TNFA −238/−308 polymorphisms gives no significant contribution to the susceptibility to schizophrenia, and is not associated with clinical variables, antipsychotic treatment response and psychopathological features, except for family history of disease, at least in the Korean population.     

Preliminary investigations on melatonin and 5-methoxytryptophol synthesis in the pineal,retina, and Harderian gland of the mole rat and in the pineal of the mouse “eyeless”

Summary Hydroxyindole-O-methyltransferase (HIOMT) activity for the synthesis of melatonin and 5-methoxytryptophol, both 5-methoxyindoles, was measured in the pineal, the Harderian gland and the retina of the mole rat and in the pineal of the mouse eyeless. In the pineal and the Harderian gland of the mole rat a larger amount of 5-methoxytryptophol than of melatonin is synthesized. 5-Methoxyindole synthesis is extremely high in the Harderian gland, whereas in the retina HIOMT activity is low and variable. In the pineal of the mouse eyeless, a low 5-methoxyindole synthesis showing no circadian rhythm is demonstrated. It is concluded that, besides the generally accepted regulation of the indole metabolism by light, in species with atrophied eyes having Harderian glands (mole rat) and in species without eyes other factors than light might be responsible for the indole metabolism in the pineal gland.     

Noradrenaline acting at central β-adrenoceptors induces interleukin-10 and suppressor of cytokine signaling-3 expression in rat brain: Implications for neurodegeneration

Evidence indicates that the monoamine neurotransmitter noradrenaline elicits anti-inflammatory actions in the central nervous system (CNS), and consequently may play a neuroprotective role where inflammatory events contribute to CNS pathology. Here we examined the ability of pharmacologically enhancing central noradrenergic tone to induce expression of anti-inflammatory cytokines in rat brain. Administration of the noradrenaline reuptake inhibitor reboxetine (15 mg/kg; ip) combined with the α₂-adrenoceptor antagonist idazoxan (1 mg/kg; ip) induced interleukin-10 (IL-10) expression in rat cortex and hippocampus. In addition, these drug treatments induced IL-10 signaling as indicated by increased STAT3 phosphorylation and suppressor of cytokine signaling-3 (SOCS-3) mRNA expression. In contrast to the profound increase in IL-10 induced by the reboxetine/idazoxan combination, the other two broad spectrum anti-inflammatory cytokines IL-4 and TGF-β were not induced by this treatment. The ability of combined treatment with reboxetine and idazoxan to induce IL-10 and SOCS3 expression was mediated by β-adrenoceptor activation, as their induction was blocked by pre-treatment with the β-adrenoceptor antagonist propranolol. Moreover, administration of the brain penetrant β₂-adrenoceptor agonist clenbuterol induced a time- and dose-dependent increase in central IL-10 and SOCS3 expression, and the ability of clenbuterol to induce IL-10 and SOCS-3 expression was blocked by the centrally acting β-adrenoceptor antagonist, propranolol, and was mimicked by the highly selective β₂-adrenoceptor agonist formoterol. In all, these data indicate that increasing central noradrenergic tone induces IL-10 production and signaling in the CNS, which may protect against neurodegeneration.     

Dietary uridine-5′-monophosphate supplementation increases potassium-evoked dopamine release and promotes neurite outgrowth in aged rats

Membrane phospholipids like phosphatidylcholine (PC) are required for cellular growth and repair, and specifically for synaptic function. PC synthesis is controlled by cellular levels of its precursor, cytidine-5'-diphosphate choline (CDP-choline), which is produced from cytidine triphosphate (CTP) and phosphocholine. In rat PC12 cells exogenous uridine was shown to elevate intracellular CDP-choline levels, by promoting the synthesis of uridine triphosphate (UTP), which was partly converted to CTP. In such cells uridine also enhanced the neurite outgrowth produced by nerve growth factor (NGF). The present study assessed the effect of dietary supplementation with uridine-5'-monophosphate disodium (UMP-2Na+, an additive in infant milk formulas) on striatal dopamine (DA) release in aged rats. Male Fischer 344 rats consumed either a control diet or one fortified with 2.5% UMP for 6 wk, ad libitum. In vivo microdialysis was then used to measure spontaneous and potassium (K+)-evoked DA release in the right striatum. Potassium (K+)-evoked DA release was significantly greater among UMP-treated rats, i.e., 341+/-21% of basal levels vs. 283+/-9% of basal levels in control rats (p<0.05); basal DA release was unchanged. In general, each animal's K+-evoked DA release correlated with its striatal DA content, measured postmortem. The levels of neurofilament-70 and neurofilament-M proteins, biomarkers of neurite outgrowth, increased to 182+/-25% (p<0.05) and 221+/-34% (p<0.01) of control values, respectively, with UMP consumption. Hence, UMP treatment not only enhances membrane phosphatide production but also can modulate two membrane-dependent processes, neurotransmitter release and neurite outgrowth, in vivo.     

Developmental expression and distribution of GABA(A) receptor α1-, α3- and β2-subunits in pig brain

The principal function of the γ-aminobutyric acid (GABA) system in the adult brain is inhibition; however, in the neonatal brain, GABA provides much of the excitatory drive. As the brain develops, transmembrane chloride gradients change and the inhibitory role of GABA is initiated and continues throughout juvenile and adult life. Previous studies have shown that GABA(A) receptor subunit expression is developmentally regulated, and it is thought that the change in GABA function from excitation to inhibition corresponds to the changeover in expression of 'immature' to 'mature' subunit isoforms. We examined the protein expression pattern and distribution of GABA type A (GABA(A)) receptor α1-, α3- and β2-subunits in the parietal cortex and hippocampus of the developing piglet brain. Four perinatal ages were studied; 14 days preterm (P-14), 10 days preterm (P-10), day of birth (P0) and at postnatal day 7 (P7). Animals were obtained by either caesarean section or spontaneous birth. Protein expression levels and subunit localization were analysed by Western blotting and immunohistochemistry, respectively. In the cortex and hippocampus, GABA(A) receptor α1-subunit showed greatest expression at P7 when compared to all other age groups (p < 0.05). In contrast, α3 expression in the cortex was elevated in preterm brain, peaking at P0, followed by a significant reduction by P7 (p < 0.05); a similar trend was observed in the hippocampus. GABA(A) receptor β2-subunit protein expression appeared relatively constant across all time points studied in both the cortex and hippocampus. Immunolabelling of the α1-, α3- and β2-subunits was observed throughout all cortical layers at every age. GABA(A) receptor α3-subunit appeared to show specific localization to layers V and VI whilst labelling for the β2-subunit was observed in layer IV. In the hippocampus of all animals, the α1- and β2-subunits were shown to immunolabel various cells and processes in the dentate gyrus (DG), CA1 and CA3; the α3-subunit was barely observed except at the stratum moleculare of the DG. We report for the first time the ontogenesis of GABA(A) receptor subunits α1, α3 and β2 in the perinatal pig brain.     

Functional expression of adrenergic and opioid receptors in Xenopus oocytes: interaction between α2- and β2-adrenergic receptors

We functionally expressed α₂-adrenergic, β₂-adrenergic, and δ-opioid receptors in Xenopus laevis oocytes. We detected receptor function as changes in currents carried by adenosine 3′,5′-cyclic monophosphate (cAMP)-regulated chloride channels provided by the cystic fibrosis transmembrane conductance regulator (CFTR) and recorded by two-electrode voltage clamp. Co-application of forskolin and isobutylmethylxanthine (IBMX) or IBMX alone produced currents with a reversal potential indicative of chloride ions only in oocytes previously injected with mRNA encoding CFTR. Isoproterenol produced concentration-dependent responses in oocytes injected with mRNA encoding β₂-adrenergic receptors and CFTR, and co-administration of propranolol antagonized these responses. Similarly, the α₂-adrenergic agonist UK14304 increased IBMX-induced currents only in oocytes injected with mRNA encoding α₂-adrenergic receptors and CFTR, and idazoxan antagonized these enhancements. The δ-opioid agonist DADLE produced concentration-related, naloxonc-reversible increases in IBMX- and forskolin-induced currents only in oocytes injected with mRNA encoding δ-opioid receptors and CFTR. In oocytes co-injected with α₂, β₂, and CFTR mRNAs, isobolographic analysis revealed an additive interaction between α₂ - and β₂-adrenergic receptors. These studies establish the oocyte as a cell system for studying the interactions among cAMP-modulating G protein-coupled receptors and provide another example of alternative coupling of α₂-adrenergic and δ-opioid receptors to G proteins, possibly G_s proteins, other than G_i proteins.     

Expression of α-, β-, and γ-synuclein in glial tumors and medulloblastomas

alpha-, beta- and gamma-synuclein are highly homologous proteins that are found predominantly in neurons. Abnormal accumulation of synucleins has been associated with diseases of the central nervous system particularly Parkinson's disease. Immunoreactivity of alpha-synuclein is demonstrated in brain tumors with neuronal differentiation and in schwannomas, whereas gamma-synuclein has been demonstrated in breast and ovarian carcinomas. The immunoreactivity of synucleins has not been described in glial tumors. Immunoreactivity of synucleins in glial cells in culture and in pathological conditions, however, suggests that synucleins may be expressed by glial tumors. We studied the expression of alpha-, beta-, and gamma-synuclein in 84 human brain tumors (24 ependymomas, 31 astrocytomas, 8 oligodendrogliomas, and 21 medulloblastomas) by immunohistochemistry. Our study demonstrates immunoreactivity for gamma-synuclein in high-grade glial tumors; immunoreactivity is found in all anaplastic ependymomas but in only 33% of ependymomas and 16% of myxopapillary ependymomas. Immunoreactivity for gamma-synuclein is noted in 63% of glioblastomas but not in other astrocytic tumors. Of medulloblastomas, 76% have immunoreactivity for either alpha- or beta-synuclein or both; no immunoreactivity for gamma-synuclein is seen in medulloblastomas.     

Down-regulation of dopamine D-2, 5-HT2 receptors and β-adrenoceptors in rat brain after prolonged treatment with a new potential antidepressant,Lu 19-005

Summary Lu 19-005 is a new phenylindan derivative with strong and equipotent inhibitory effect on dopamine (DA), noradrenaline (NA) and serotonin (5-HT) uptake. The adaptive effects of 2 weeks treatment with Lu 19-005, on receptor bindingin vitro and on d-amphetamine responsivenessin vivo have been investigated in rats. One or 3 days after the final dose the number of-adrenoceptors and of 5-HT₂ and DA D-2 receptors was decreased by 20–30%, whereas ₁-adrenoceptor number was slightly decreased only 1 day after withdrawal. The DA D-2 receptor number remained decreased at 7 days withdrawal, but returned to normal after another 3 days. The brain levels of DA, NA and 5-HT were not changed by 2 weeks' Lu 19-005 treatment. The down-regulation of DA D-2 receptors was accompanied by tolerance to d-amphetamine-induced hypermotility (after low doses) and stereotyped licking or biting (after a high dose). The tolerance to d-amphetamineinduced hypermotility was maximal at 3–5 days withdrawal time, and remained significant also 15 days after the last dose. An acute dose of Lu 19-005 did not modify the effects of d-amphetamine. The results are discussed in relation to the effect of prolonged treatment with other antidepressant drugs.On leave from Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, PL-31-343 Krakow, Poland.     

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Background

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.

Methods

In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.

Results

Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.

Conclusions

Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.