Changes In Binding Parameters Research Articles

In Vitro Investigation of the Interaction of Tolbutamide and Losartan with Human Serum Albumin in Hyperglycemia States.

Serum albumin is exposed to numerous structural modifications which affect its stability and activity. Glycation is one of the processes leading to the loss of the original properties of the albumin and physiological function disorder. In terms of long lasting states of the hyperglycemia, Advanced Glycation End-products (AGEs) are formed. AGEs are responsible for cellular and tissue structure damage that cause the appearance of a number of health consequences and premature aging. The aim of the present study was to analyze the conformational changes of serum albumin by glycation—“fructation”—using multiple spectroscopic techniques, such as absorption (UV-Vis), fluorescence (SFM), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy and evaluate of possible alteration of binding and competition between tolbutamide (TB, a first-generation sulfonylurea oral hypoglycemic drug) and losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension (1st line with a coexisting diabetes)) in binding to non-glycated (HSA) and glycated (gHSAFRC) human serum albumin in high-affinity binding sites. The studies allowed us to indicate the structural alterations of human serum albumin as a result of fructose glycation. Changes in binding parameters, such as association or Stern-Volmer constants suggest that glycation increases the affinity of TB and LOS towards albumin and affects interactions between them. The process of albumin glycation influences the pharmacokinetics of drugs, thus monitored pharmacotherapy is reasonable in the case of diabetes and hypertension polypharmacy. This information may lead to the development of more effective drug treatments based on personalized medicine for patients with diabetes. Our studies suggest the validity of monitored polypharmacy of diabetes and coexisting diseases.

Epitope mapping of monoclonal antibody HPT-101: a study combining dynamic force spectroscopy, ELISA and molecular dynamics simulations

By combining enzyme-linked immunosorbent assay (ELISA) and optical tweezers-assisted dynamic force spectroscopy (DFS), we identify for the first time the binding epitope of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to the Alzheimer’s disease relevant peptide tau[pThr231/pSer235] on the level of single amino acids. In particular, seven tau isoforms are synthesized by replacing binding relevant amino acids by a neutral alanine (alanine scanning). From the binding between mAb HPT-101 and the alanine-scan derivatives, we extract specific binding parameters such as bond lifetime , binding length , free energy of activation (DFS) and affinity constant (ELISA, DFS). Based on these quantities, we propose criteria to identify essential, secondary and non-essential amino acids, being representative of the antibody binding epitope. The obtained results are found to be in full accord for both experimental techniques. In order to elucidate the microscopic origin of the change in binding parameters, we perform molecular dynamics (MD) simulations of the free epitope in solution for both its parent and modified form. By taking the end-to-end distance and the distance between the α-carbons of the phosphorylated residues as gauging parameters, we measure how the structure of the epitope depends on the type of substitution. In particular, whereas is sometimes conserved between the parent and modified form, strongly changes depending on the type of substitution, correlating well with the experimental data. These results are highly significant, offering a detailed microscopic picture of molecular recognition.

Physical exercise improves synaptic dysfunction and recovers the loss of survival factors in 3xTg-AD mouse brain

Physical exercise has become a potentially beneficial therapy for reducing neurodegeneration symptoms in Alzheimer's disease. Previous studies have shown that cognitive deterioration, anxiety and the startle response observed in 7-month-old 3xTg-AD mice were ameliorated after 6 months of free access to a running wheel. Also, alterations in synaptic response to paired-pulse stimulation were improved. The present study further investigated some molecular mechanisms underlying the beneficial effects of 6 months of voluntary exercise on synaptic plasticity in 7-month-old 3xTg-AD mice. Changes in binding parameters of [3H]-flunitrazepam to GABAA receptor and of [3H]-MK-801 to NMDA receptor in cerebral cortex of 3xTgAD mice were restored by voluntary exercise. In addition, reduced expression levels of NMDA receptor NR2B subunit were reestablished. The synaptic proteins synaptophysin and PSD-95 and the neuroprotective proteins GDNF and SIRT1 were downregulated in 3xTgAD mice and were recovered by exercise treatment. Overall, in this paper we highlight the fact that different interrelated mechanisms are involved in the beneficial effects of exercise on synaptic plasticity alterations in the 3xTg-AD mouse model.

Baclofen reestablishes micro-opioid receptor levels modified by morphine withdrawal syndrome in either sex.

We have previously shown that the GABA(B) agonist baclofen (BAC) prevents the expression of morphine (MOR) withdrawal syndrome in male as well as female mice. In addition, we have demonstrated that BAC reestablishes the dopamine levels modified by MOR withdrawal syndrome in male mice. The aim of the present study was to evaluate the micro-opioid receptor binding parameters in striatum and frontal cortex of male and female mice during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice of either sex were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the tenth day, dependent animals received naloxone (NAL) (6 mg/kg, i.p.) 60 min after the last dose of MOR and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL injection. Thirty min after NAL or saline injection mice were sacrificed, brains were collected, and the striatum and frontal cortex were dissected in order to perform binding studies with [(3)H][DAMGO]. The density of micro-opioid receptor increased significantly during MOR withdrawal in male and female striatum as well as in male cortex. In addition, in both brain areas the B(max) was higher in male than in female mice during MOR withdrawal. Finally, BAC pretreatment of MOR withdrawn mice reestablished the levels of micro-opioid receptor by significantly decreasing the B(max) in either sex. In conclusion, although there were sex differences in the micro-opioid receptor density during MOR withdrawal syndrome, BAC was able to reestablish the changes in binding parameters induced by the NAL-precipitated withdrawal in female and male mice.

ACUTE CHANGES IN 3H-PAC AND 125I-PYY BINDING IN THE NUCLEUS TRACTUS SOLITARII AND HYPOTHALAMUS AFTER A HYPERTENSIVE STIMULUS

Activation of alpha-2-adrenergic and neuropeptide Y (NPY) receptors in the nucleus tractus solitarii (NTS) induces hypotension and bradycardia. On the contrary, activation of angiotensin II (Ang II) receptors leads to hypertension. Acute changes in binding parameters of alpha-2-adrenergic, NPY and Ang II receptors were evaluated in the NTS and paraventricular hypothalamic nucleus (PVN) of rats after a hypertensive stimulus employing quantitative receptor autoradiography. Saturation experiments showed a decrease in the number (Bmax) of alpha-2-adrenergic binding sites in the NTS 6 hours after coarctation-induced hypertension. Furthermore, the affinity of NPY receptors was diminished as seen by the increase in the KD value of 125 I-PYY. Tyrosine hydroxylase and NPY immunoreactivities were increased in the NTS and ventral medulla. Binding of 125 I-Ang II was not changed in the NTS. Binding of all ligands analyzed was not altered in the PVN. The results suggest an acute down-regulation of alpha-2-adrenergic and NPY receptors involved with hypotension in response to hypertensive stimulus, which might be related to an increased availability of catecholamines and NPY in the NTS.

Increased density of M1 receptors in the hippocampus of juvenile rats chronically deprived of NGF

Binding studies were used to assess the changes in affinity and/or number of M1 muscarinic receptors in hippocampi from juvenile rats chronically deprived of NGF. NGF deprivation was obtained by implanting into right ventricle at postnatal day 2 (P2) hybrydoma cells secreting high levels of monoclonal antibodies against NGF (αD11). Parenteral myeloma cells (P3U) were used as controls. Competition experiments were used to characterise the [ 3 H ]-PNZ binding sites in membrane preparations of hippocampi from rats sacrificed at P15. [ 3 H ]-PNZ bound M1 receptors both in P3U and αD11 group as shown by displacing potency order of antagonists: TLZ=4-DAMP>PNZ> p-F-HHSiD>MTC. The deprivation of NGF for two weeks significantly increased the number of M1 receptors without changing the K i values of antagonists with exception of methoctramine which showed an increase in affinity in αD11 group. Similar changes in binding parameters were already observed after the first week of anti-NGF treatment. In contrast, a treatment for a week with implant at postnatal day 15 failed to produce any changes in M1 binding parameters. These results provide further physiological evidence for developmentally regulated modulatory role of NGF in the cholinergic function in the hippocampus.

Influence of lipid composition on pediocin PA-1 binding to phospholipid vesicles.

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.

Reduction in number of dopamine uptake sites but unchanged number of piperazine-acceptor/CYP450IID6 binding sites in the human caudate nucleus in aging

A substantial decrease in number of striatal dopamine uptake sites is a characteristic finding in aging. This decrease resembles the dopaminergic nigro-striatal degeneration in Parkinson's disease (PD). A dysfunction of cytochrome P450IID6 (debrisoquine-4-hydroxylase) is suggested to be involved in the pathogenesis of PD. In this study, binding sites associated with the neuronal form of P450IID6 were studied in the caudate nucleus from individuals in the age range 20–81 years using [ 3H]GBR 12935 as a radioligand. No significant changes in binding parameters were obtained, while in the same region a significant decrease in number of dopamine uptake sites occurred. Thus, in aging, P450IID6 and dopaminergic degeneration seem not to be functionally related in this region. Whether such a relation exists in PD is still to be examined.

Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

Proton inhibition of [ 3H]resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord

Protons and capsaicin activate overlapping subsets of sensory nerves by opening ion conductances of similar properties. We have used the [ 3H]resiniferatoxin binding assay utilizing rat spinal cord membranes to elucidate the possible interaction of protons at the vanilloid (capsaicin) receptor. Using low pH (pH 6.0 and pH 5.0) buffers, a time-dependent gradual decrease was observed in specific resiniferatoxin binding. Protons inhibited resiniferatoxin binding with an IC 50 of pH 5.3 ± 0.1. In experiments in which the concentration of [ 3H]resiniferatoxin was varied, protons reduced the max value by approximately 40% with a corresponding 2-fold decrease in affinity. No change however, was observed in binding cooperativity (the Hill coefficients were 1.7 ± 0.1 and 1.6 ± 0.2 in the presence of pH 7.4 and pH 5.0 buffers, respectively). These changes in binding parameters are consist with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity ( K i=5.0 ± 1.0 μM) very similar to that determined in the presence of a pH 7.4 buffer ( K i = 3.0 ± 1.5 μM). A cyclooxygenase inhibitor, indomethacin (up to 10 μM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I 2 and E 1, both at 100 μM). We conclude that protons interact at vanilloid receptors in the rat spinal cord; this interaction is either non-competitive or mixed in nature, and probably is not related to prostanoid generation. Protons and/or putative proton-generated mediators might represent endogenous modulators of the vanilloid receptor.

2-[125I]iodomelatonin binding sites in murine mammary tissue.

2-[125I]iodomelatonin binds in vitro to murine mammary gland membranes, in a specific, saturable, reversible and temperature-dependent and pH-dependent way, displaying an affinity within the low nM range. The order of affinity to displace the radioligand from mammary membranes was: 6-hydroxy-melatonin > 5-hydroxyindoleamines > noradrenaline > 6-chloromelatonin > 5-methoxyindoles (melatonin included). There was a diurnal rhythm in Kd and Bmax of 2-[125I]iodomelatonin binding, displaying the highest affinity and the lowest binding site concentration during the scotophase. The functional significance of melatonin binding sites was indicated by changes in binding parameters related to different rates of mammary growth during puberty, estrous cycle, pregnancy, lactation and the postlactation period.

Intrathecal baclofen down-regulates GABAB receptors in the rat substantia gelatinosa

At present, it is not clear why drug tolerance develops in patients receiving intrathecal baclofen for the chronic treatment of spasticity of spinal origin. To investigate the mechanisms of tolerance to the gamma-aminobutyric acid (GABAB) agonist baclofen, rats were implanted with intrathecal catheters and continuously infused with either the drug or saline for 1, 3, or 7 days. The dose chosen, 1 microgram/hr, initially caused profound hindlimb motor weakness, but by Day 7 the rats had adapted to the drug and exhibited only minimal motor impairment. The animals were sacrificed on Day 1, 3, or 7 and quantitative autoradiography was used to determine the binding density and affinity of the GABAB receptors in the substantia gelatinosa of the lumbar spinal cord. After 1 day of drug infusion there was no change in binding parameters, but after 3 and especially after 7 days there was a significant decrease in the GABAB binding density (74% and 66%, respectively) in baclofen-treated rats as compared to saline-treated control rats. This GABAB receptor down-regulation correlated with tolerance to the motor weakness in the baclofen-treated animals and suggests that similar mechanisms contribute to drug tolerance in patients.

GABAergic Control of Receptivity in the Female Rat

GABAergic neurotransmission has been implicated in the control of the steroid-dependent behavior, lordosis. GABA has dual effects on lordosis: it facilitates lordosis through actions in the medial hypothalamus (mHYP) and it inhibits lordosis through actions in the preoptic area (POA). In the present study, gonadally intact and ovariectomized female rats were behaviorally tested with a sexually active male. Brains were removed from sexually receptive female either 1 or 24 h after behavioral testing. There was a significant difference in endogenous GABA concentration in HYP and POA between receptive, postreceptive and ovariectomized nonreceptive females. Specifically, GABA levels in postreceptive females were higher in the HYP (20%) and lower in the POA (21%) in comparison to receptive females (p less than 0.05). There was also a significant change in binding parameters of 3H-muscimol in the HYP and POA of receptive females as compared to 24 h postreceptive and ovariectomized rats. Attempts to modulate 3H-GABA release from hypothalamic tissue slices by estrogen or progesterone in ovariectomized rats yielded no effect on this parameter.

Thyroid Hormone Regulates Rat Pituitary Insulin-Like Growth Factor-I Receptors

As we previously obtained evidence that insulin-like growth factor-I (IGF-I) inhibits T3-induced GH secretion and GH mRNA expression without affecting basal GH secretion in thyroidectomized rat pituitary cells grown in hypothyroid medium, we examined changes in IGF-I receptors in the pituitary gland, as induced by thyroid hormone. Thyroidectomized rats and a quantitative receptor autoradiographic method were used. The density of [125I]IGF-I-binding sites in the anterior pituitary gland decreased 4 weeks after thyroidectomy; that is a significant decrease in the number of the receptors compared to findings in control rats (P less than 0.01). The affinity (Kd) remained unchanged. There were no changes in binding parameters in the ventroposterior thalamic nucleus in the brain, renal cortex, and liver parenchyma. The ip administration of T4 once a day (48 micrograms/kg) for 1-2 weeks compensated for the decrease in the binding capacity of [125I]IGF-I-binding sites to that of the control values (P less than 0.01). We propose that IGF-I receptors in the anterior pituitary gland may be regulated by thyroid hormone.

Factors causing age-dependent changes in phenytoin tissue and serum binding in rats.

The factors that cause age-dependent changes in phenytoin tissue and serum protein binding in rats were studied. It was confirmed that the age-dependent changes in the concentration of tissue constituents to which phenytoin bound mainly governed the change in phenytoin tissue binding. The concentration of the tissue constituents, protein and phospholipid, was changed by the water content in tissues in the growth process of rats. The increase in serum protein binding of phenytoin in the growth process of rats was caused by changes in both serum albumin concentration and binding parameters of phenytoin to serum albumin. The changes in binding parameters of phenytoin to serum albumin were led by those in the molar rations of free fatty acids to albumin.

Unexpectedly high levels of opioid peptides in rat brain membranes

Several considerations led us to directly measure, using radioimmunoassy, the levels of dynorphin-A(1–8), cholecystokinin-8, and Met 5-enkephalin-Arg 6-Gly 7-Leu 8 (MERGL) in lysed-P2 membranes, membranes incubated 60 min at 25°C in 50 mM TRIS-HCl at pH 7.4, and membranes preincubated 60 min at 25°C in TRIS buffer containing 0.4 M NaCl. Lysed-P2 membranes contained neuropeptides at levels between 0.253 and 1.727 pmol/mg protein. Incubating membranes in buffer alone decreased membrane peptide levels by 40 to 60%. Preincubations in the presence of 0.4 M NaCl resulted in even greater reductions in the peptide content of membranes (between 58 and 90%), without changing [ 3H]etorphine binding. Detailed comparisons of the two binding sites labeled by [ 3H]naloxone revealed changes in binding parameters induced by preincubation with NaCl. Taken collectively, these studies demonstrate that lysed-P2 membranes contain sequestered peptides that are not bound to opiate receptors.

Acute cold-restraint stress affects α2-adrenoceptors in specific brain regions of the rat

The effect of acute cold-restraint stress on binding of [ 3H]rauwolscine to α 2-adrenoceptors was investigated in 10 regions of rat brain. Acute stress: (1) significantly decreased the density but had no significant effect on the affinity of binding sites in the hippocampus; (2) decreased density and increased affinity in the amygdala; and (3) increased density and decreased the affinity in the midbrain. Seven other brain regions showed no significant changes in binding parameters in response to stress. These results, together with previous findings in this laboratory showed that alteration by restraint stress of noradrenaline levels in amygdala and hippocampus, but not other regions, indicate an association between neurotransmitter turnover and receptor function.

Calcium discriminates two [ 3H]kainate binding sites with different molecular target sizes in rat cortex

Binding of [ 3H]kainate to the kainate subtype of excitatory amino acid receptors revealed two binding sites. The high-affinity site ( K d =3.5 nM) was sensitive to calcium ions and has a molecular target size as determined by high-energy radiation inactivation analysis of 76,600 daltons. The low-affinity site ( K d =65 nM) was insensitive to calcium ions and has a molecular target size similar to the quisqualate-subtype of excitatory amino acid receptors. The change in binding parameters of the two sites with radiation dose strongly suggests that low affinity, calcium-insensitive [ 3H]kainate binding sites are equivalent to quisqualate sites.

Selective destruction of the serotonergic fibers of the fornix-fimbria and cingulum bundle increases 5-HT 1 but not 5-HT 2 receptors in rat midbrain

Selected and localized lesions of serotonergic (5-HT) neurons were made by microinjection of 5,7-dihydroxytryptamine (5,7-DHT), after pretreatment with desipramine, into the cingulum bundle and fornix-fimbria; these are the major serotonergic hippocampal inputs from the median raphe nucleus. Two weeks after the lesion, the binding of [ 3H]5-HT (5-HT 1 receptor) was determined in the hippocampus which receives the afferent terminals and, in addition, in the septum/hypothalamus and midbrain from where the fibers originate. Scatchard analysis showed there was no significant change in binding parameters in the hippocampus; however, a significant increase was observed in the B max in the midbrain (38%) with no change in the K D. The caudate which receives 5-HT inputs via other 5-HT tracts was not affected by the lesion. The changes in 5-HT 1 receptor number or affinity were not observed 6 days or 5 weeks after the lesion. The binding of the ligands [ 3H]spiroperidol and [ 3H]ketanserin to the 5-HT 2 receptor population was also determined in the same brain areas; no changes in receptor binding occurred two weeks after the lesion. These experiments demonstrate that a selective lesion of the serotonergic system can increase 5-HT 1 receptors in the midbrain, which contains the serotonin cell bodies. In addition, as 5-HT 2 binding is not altered, this further supports the hypothesis that 5-HT 1 and 5-HT 2 receptors are distinct populations of receptors.

The Effect of Drugs on Albumin Binding of Bilirubin and on Free Bilirubin Concentration as Determined by Fluorescence Quenching

Fluorescence quenching was used to determine the influence of five drugs commonly used in the newborn infant, on the binding of bilirubin to albumin, and to quantitate the resultant changes in binding parameters and the free bilirubin concentration. The five drugs studied were digoxin, furosemide, diazepam, phenytoin sodium, and theophylline. The primary influence of digoxin, furosemide, diazepam, and phenytoin sodium was on the affinity of albumin to bind bilirubin. They also affected the capacity of albumin to bind bilirubin, but to a lesser extent. Furosemide reduced the apparent binding affinity by 60 percent (p = 0.01), phenytoin sodium by 58 percent (p < 0.01), digoxin by 52 percent (p = 0.02), and diazepam by 45 percent (p = 0.03). Furosemide reduced the apparent binding capacity by 24 percent (p < 0.001), digoxin by 23 percent (p = 0.04), and diazepam by 13 percent (p = 0.10). Phenytoin sodium had a different effect on the apparent binding capacity: It increased the apparent capacity by 30 percent (p < 0.01). Theophylline had no significant effects on either binding affinity or capacity or free bilirubin concentration. Furosemide, digoxin, and diazepam significantly increased free bilirubin concentration at all total bilirubin concentrations; therefore, these drugs should be used with caution in the jaundiced newborn infant. Phenytoin sodium increased free bilirubin concentrations in the low total bilirubin ranges only; however, the increase was marginal and unlikely to be clinically significant.