Real-time RT-PCR threshold cycles value for Kir6.1 from the blood correlates with parameters of vascular function: a potential for the vascular function biomarker?

Abstract We examined the presence of KATP channel subunits, Kir6.1 and SUR2B, mRNAs in the blood and vascular function in healthy volunteers (41 males, 34 females). Real-time reverse transcriptase (RT)-PCR threshold cycles (Ct) was used as an indicator of mRNA levels. Baseline skin perfusion and the post-occlusion reactive hyperemia response exhibited a significant positive correlation with Ct for Kir6.1. There was no correlation between Kir6.1 Ct and brachial artery flow-mediated dilatation. Gender had no influence on relationships between blood Kir6.1 Ct and vascular function. We conclude that blood Kir6.1 mRNA levels could be potentially used as a biomarker of the vascular function.

Impact of plasma-protein binding on receptor occupancy: an analytical description.

In this paper we analyse the dynamics of an inhibitor I which can either bind to a receptor R or to a plasma protein P. Assuming typical association and dissociation rates, we find that after an initial dose of inhibitor, there are three time scales: a short one, measured in fractions of seconds, in which the inhibitor concentration and the plasma-protein complex jump to quasi-stationary values, a medium one, measured in seconds in which the receptor complex rises to an equilibrium value and a large one, measured in hours in which the inhibitor-receptor complex slowly drops down to zero. We show that the average receptor occupancy, the pharmacologically relevant quantity, taken over, say, 24h reaches a maximal value for a specific value of the plasma-protein binding constant. Potentially, understanding and exploiting this optimum could be of great interest to those involved in drug discovery and development.

Impact of the sulfonylurea receptor 1 (SUR1) exon 16-3c/t polymorphism on acute hyperglycaemia in type 2 diabetic patients.

Epidemiological data collected over the last few decades have demonstrated the significant role of acute (especially postprandial) hyperglycaemia in the development of macrovascular complications in patients with type 2 diabetes. However, the influence of SUR1 exon 16-3c/t polymorphism on impaired insulin secretion during acute hyperglycaemic episodes has not yet been evaluated. We studied 40 type 2 diabetic patients. Single nucleotide polymorphism in the sulfonylurea receptor gene was examined by means of PCR-RLFP. In every patient, fasting insulin, proinsulin, C-peptide and 1,5-anhydro-d-glucitol concentrations were assayed as markers of insulin secretion, peripheral resistance to insulin, and acute hyperglycaemia. The distribution of SUR1 exon 16-3c/t polymorphism was tt 35%, tc -40%, and cc -25%. By means of analysis of covariance, it was revealed that 1,5-anhydro-d-glucitol plasma levels are associated with SUR1 exon 16-3c/t polymorphism. However, the HOMA(IR) score influenced 1,5-anhydro-d-glucitol levels in plasma at a higher level of statistical power than the genetic variant. Our results suggest that SUR1 exon 16-3c/t polymorphism is only a partial determinant of acute hyperglycaemia-cardiovascular risk factor in type 2 diabetes.

[Study on the relationship between sulfonylurea receptor 1 gene polymorphism and type 2 diabetes mellitus].

To study whether the 3c/t polymorphism of the sulfonylurea receptor 1 (SUR1) gene exon16 increased the risk of type 2 diabetes mellitus in type 2 diabetes mellitus pedigrees in Han population in south area of China. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used in 46 type 2 diabetes mellitus pedigrees. The polymorphism in SUR1 was tested and analyzed by Mantel-Haenszel chi(2) test. Frequencies of SUR1-3c/t polymorphism had no significant difference between type 2 diabetes mellitus and normal relatives (genotypes chi(2)=3.224, P=0.199; frequency of allele chi(2)=1.250, P=0.264). In all subjects, type 2 diabetes mellitus and normal relatives, SUR1-3c/t genotypes were listed (cc: 29.3%, 30.2%, 28.3%; ct: 50.7%, 53.8%, 47.2%; tt: 20%, 16.0%, 24.5% respectively). The frequencies of c were 54.7%, 57.1% and 51.9% respectively. The frequency of c is lower than Han population in northern China. The results show that SUR1 exon16-3c/t polymorphism is not associated with type 2 diabetes mellitus in the population.

Decreased platelet [3H]paroxetine binding sites in suicide attempters.

Research to date would suggest the possible involvement of the serotonin (5-HT) system in the pathophysiology of suicide. With this study, we aimed to investigate the platelet 5-HT transporter, by means of the specific binding of tritiated paroxetine ([3H]Par), in a sample of 20 suicide attempters recruited at a first-aid service, as compared with healthy control subjects and psychiatric patients with no current or previous history of suicide attempt. The results, showing a decreased number of [3H]Par binding sites in suicide attempters, would suggest the involvement of the presynaptic 5-HT transporter in self-aggressive behavior.

Effect of bupropion on immunodensity of putative imidazoline receptors on platelets of depressed patients.

A substantial number of studies have demonstrated increased imidazoline receptors (I1 binding sites) on platelets of depressed patients and downregulation following antidepressant treatments. Herein, imidazoline receptor binding protein (IRBP) antiserum was used to quantify imidazoline receptors on platelets of depressed patients before and after treatment with the atypical aminoketone antidepressant, bupropion. Western blots revealed an increase in IRBP-immunodensity (p = 0.01, two-tailed) in a 33 kDa protein band in untreated depressed patients (n = 21) as compared with controls (n = 17). This band has been positively correlated with I1 binding sites on platelets. Following 6 weeks' treatment with bupropion, IRBP-immunodensity was downregulated in depressed patients (p = 0.03, paired t-test); predominantly in responders (p = 0.005). Patients non-responsive to bupropion (n = 5) were significantly different from responders (p = 0.05) by exhibiting no elevation in IRBP-immunodensity at pre-treatment and no downregulation of the 33 kDa band after treatment. IRBP-immunodensity was negatively correlated (r = -0.79, p = 0.01) with plasma concentrations of bupropion and its metabolites at week-4 of BUP treatment. Thus, a 33-kDa IRBP on platelet plasma membranes is elevated in depression and normalized in responders to bupropion.

Plasma agmatine and platelet imidazoline receptors in depression.

Plasma agmatine concentrations are elevated significantly in depressed patients compared to healthy controls. Treatment with the antidepressant bupropion normalized plasma agmatine levels. Correlational evidence is presented that a change in plasma agmatine levels may lead to similar changes in platelet I1 imidazoline receptors.

Expression of cannabinoid receptors and their gene transcripts in human blood cells.

1. This study shows that the human cannabinoid receptors and their gene transcripts can be analyzed in blood samples when combined with polymerase chain reaction. The results also demonstrate that the expression of the cannabinoid receptors is dependent on gender and ethnic background. 2. Normal human volunteers who do not use marijuana have genes that encode for the marijuana (cannabinoid) receptor proteins. 3. Primer pairs from CB1 and CB2 cDNA coding region sequences showed identical amplified DNA band sizes in both DNA-PCR and reverse PCR, with human templates. This suggests that the CB1 and CB2 genes are intronless at least in their coding regions. 4. An advantage of the coding region being intronless may be that the expression of these genes will have one major RNA processing event to skip, thus making the conditions of their expression relatively quick and simple. This advantage may have implications related to the biological functions of these proteins. 5. We therefore concluded that the existence of human cannabinoid receptors and genes along with the discovery of endogenous cannabinoids (endocannabinoids) may be useful markers in elucidating the role(s) and mechanism(s) of action of cannabinoids.

Clinical characterization of polymorphisms in the sulphonylurea receptor 1 gene in Japanese subjects with Type 2 diabetes mellitus.

To assess the association of polymorphisms at the sulphonylurea receptor (SUR1) gene with the development of Type 2 diabetes mellitus, 456 subjects, 236 with Type 2 diabetes and 220 non-diabetic controls, were analysed for variants at exon 7, exon 22 and intron 24 of the SUR1 gene by the polymerase chain reaction and restriction fragment length polymorphism. The T761T substitution in exon 22 of the SUR1 gene was not found in either diabetic patients or non-diabetic controls. Both the exon 7 variant and the intron 24 variant were present in both groups at similar frequencies. No significant association was seen between either variant and obesity. Diabetic patients homozygous for the -3C allele of intron 24 had a higher ratio of positive family history than patients homozygous for the -3T allele (p = 0.03). We conclude that these polymorphisms are not major determinants of diabetes and obesity in the Japanese population.

Circannual variations in the binding of [3H]lysergic acid diethylamide to serotonin2A receptors and of [3H]paroxetine to serotonin uptake sites in platelets from healthy volunteers.

BACKGROUND: Circannual variations occur in several serotonergic parameters, including platelet serotonin uptake and platelet [3H]imipramine binding. METHODS: Binding of [3H]lysergic acid diethylamide ([3H]LSD) to platelet serotonin (5-HT)2A receptors and binding of [3H]paroxetine to platelet serotonin uptake sites were studied longitudinally for 1 year in 12 healthy volunteers. RESULTS: For [3H]LSD, the number of binding sites (Bmax) showed no significant seasonal variation (two-way analysis of variance), although Bmax was significantly higher during the months October through February than during the months April through August (32.6 vs. 29.8 fmol/mg protein; p = .015). For [3H]paroxetine, Bmax showed a significant seasonal variation (p = .003) with maximum in August (1322 fmol/mg protein) and minimum in February (1168 fmol/mg protein). The affinity constant (Kd) showed a significant seasonal variation for [3H]LSD binding (p = .046), but not for [3H]paroxetine binding. The seasonal fluctuations in [3H]LSD binding and in paroxetine binding tended to be inversely correlated for Bmax (r = -.70; p = .08) and were significantly negatively correlated for Kd (r = -.88; p = .009). CONCLUSIONS: The present study demonstrates a seasonal effect on platelet serotonin uptake site binding and indicates a possible seasonal effect on 5-HT2A receptor binding. The results imply that circannual fluctuations should be taken into account when these platelet serotonin markers are studied.

Density of imidazoline receptors in platelets of euthymic patients with bipolar affective disorder and in brains of lithium-treated rats.

BACKGROUND: Platelet imidazoline receptors have been shown to be up-regulated in patients with unipolar major depression. This study examines the status of imidazoline receptor proteins in platelets of euthymic bipolar patients and in brains of lithium-treated rats. METHODS: Platelets were collected from 12 bipolar patients (lithium-treated or drug-free) and brains from chronic lithium-treated rats. Imidazoline receptors were quantitated by immunoblotting, using a specific antiserum, and/or radioligand binding. RESULTS: No changes in platelet imidazoline receptors (35-kDa and 45-kDa proteins) were found. Lithium treatment did not alter brain imidazoline receptors (29/30-kDa, 45-kDa, and 66-kDa proteins or density/affinity of [3H]-idazoxan binding sites). CONCLUSIONS: Imidazoline receptor proteins are not altered in platelets of euthymic patients with bipolar affective disorder.

Platelet imidazoline receptors and regulatory G proteins in patients with major depression.

The newly discovered imidazoline receptors have been found to be upregulated in patients with major depression (platelet 45 kDa and 35 kDa proteins) and in suicide victims (brain 45 kDa protein). The signalling pathways coupled to these receptors are not known however. The aim of this study was to quantify, in platelets of depressed patients, the density of various G proteins to assess possible associations with the abundance of imidazoline proteins. There were positive correlations between the immunoreactivities of 45 kDa imidazoline receptors and those of G alpha q/11 (r = 0.64, n = 19, p < 0.005), G alpha i2 (r = 0.46, n = 22, p < 0.05) and G beta (r = 0.62, n = 18, p < 0.01) proteins. The relationship with regulatory G alpha q/11 proteins suggests that this 45 kDa protein (putative I1 imidazoline receptor) may couple to phosphoinositide pathway in platelets. This finding might be of relevance in understanding the functional implications of the abnormal higher expression of imidazoline receptors (45 kDa protein) in the pathogenesis of major depression.

123I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB1 receptors.

We have investigated the binding of 123I-labeled N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methy l-1 H-pyrazole-3-carboxamide (AM251), an analog of the cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide] in the mouse brain. Following intravenous injection, the peak whole-brain uptake of about 1% of the administered activity occurred at about 2 h. By 8 h radioactivity in brain had declined to about half its peak value. High-performance liquid chromatographic analysis showed that > 70% of radioactivity extracted from brain at 2 h was still present as [123I]AM251. Co-injection of SR141716A inhibited the in vivo brain binding of [123I]AM251 dose dependently. At 2 mg/kg, the highest dose that could be tested, inhibition was 50% at 2 h post-administration. The ED50 value calculated assuming that 2 mg/kg gave near-maximal inhibition was about 0.1 mg/kg. In contrast to the brain, radioactivity in other major organs (blood, liver, kidney, heart and lung) was little affected by SR141716A. The regional binding of [123I]AM251 in the brain was consistent with the published distribution of cannabinoid receptors in rat brain, in that the order was hippocampus, striatum > cerebellum > brain stem. delta 9-Tetrahydrocannabinol co-administered intravenously at 10 mg/kg, a dose which induced catalepsy and decreased locomotor activity, decreased the 2 h brain uptake of [123I]AM251 by 10%, but this was not significant (P = 0.08). In in vitro binding assays with mouse hippocampal membranes, tetrahydrocannabinol inhibited binding of [123I]AM251 with an IC50 value of about 700 nM, compared with about 0.2 nM for SR141716A.

Platelet I1-imidazoline binding sites are decreased by two dissimilar antidepressant agents in depressed patients.

Previous studies have indicated that there may be a dysregulation of alpha 2-adrenoceptors and imidazoline receptors in depression. This study compares the effects of chronic antidepressant treatment with a serotonin reuptake inhibitor (fluoxetine) versus a noradrenaline reuptake inhibitor (desipramine) on the binding parameters of the platelet imidazoline binding site (subtype I1) and of the platelet alpha 2-adrenoceptor in depressed patients. After 6 weeks of treatment with either antidepressant, platelet I1 binding sites became normalized (i.e. downregulated). A negative correlation was obtained between plasma epinephrine concentrations and platelet alpha 2-adrenoceptor Bmax values within the samples, but no correlation was obtained between any plasma catecholamine and a platelet I1 binding parameter. An additional finding was the increased affinity of alpha 2-adrenoceptors for p125I-clonidine in untreated depressed patients compared to healthy subjects. Because of the density of platelet I1 binding sites was downregulated by both of the antidepressants, we postulate that a decrease in platelet I1 binding site density may be related to an improved state from depression that these antidepressants produce.

Platelet I1-imidazoline binding sites are elevated in depression but not generalized anxiety disorder.

Depressed patients have been reported to have a higher than normal density of platelet binding sites for 3H-clonidine, an alpha 2-adrenoceptor agonist. Paradoxically, other studies using 3H-alpha 2, antagonists have found no differences from controls. Because 3H-clonidine interacts with platelet alpha 2-adrenoceptors to form G-protein complexes, whereas 3H-alpha 2-antagonists bind with uncoupled receptors, an elevation in G-protein coupling might explain this paradox. Another possibility is that depression might be associated with increased non-adrenergic I1-imidazoline binding sites, which are also clonidine sensitive. To distinguish these possibilities, we utilized p125I-clonidine to measure density (Bmax) and affinity (KD) of platelet G-protein coupled alpha 2-adrenoceptors as well as platelet I1 binding sites, and compared diagnostic groups of major depressive disorder (MDD), generalized anxiety disorder (GAD) and healthy subjects. Specific inhibition of binding by norepinephrine (NE = 10 microM) was used to selectively quantify alpha 2-adrenoceptors, whereas inhibition by 10 microM moxonidine (a > 100-fold selective I1 ligand) quantified I1 binding sites under a NE mask. I1 sites were found to be markedly elevated by, on average, +136% in MDD patients (p = .0007), whereas there was only a marginal increase in alpha 2-adrenoceptor Bmax values in MDD patients (p = .08; GAD and healthy subjects did not differ). Treatment of MDD patients for 6-8 weeks with desipramine downregulated I1 sites as well as alpha 2-adrenoceptors. Positive correlations were also noted for both sites: (a) between Bmax values and the severity of depression (using the Hamilton Depression Rating Scale); and (b) between end-of-treatment plasma desipramine concentrations and the extent of downregulation in Bmax values when subject groups were pooled. None of the binding parameters was associated with plasma catecholamine concentrations. The results suggest that an increased density of platelet I1 binding sites may partially explain the utility of radiolabeled clonidine as a potential biological marker for depressive illness, although an additional increase in G-protein coupling cannot be excluded.

Erythrocyte ouabain binding and intracellular Na+ in normotensive obese women and obese women receiving medication for hypertension.

People with "primary obesity" may be hypertensive because they have lost their ability to compensate for the effect of low Na+-K+-ATPase levels on blood pressure. In obese patients receiving hypertensive medication (n = 13), but not in normotensive nonmedicated patients (n = 42), diastolic blood pressure was inversely correlated with erythrocyte ouabain binding (P less than 0.02) and directly correlated with intracellular Na+ concentration (P less than 0.01). Moreover, there was a stronger inverse relationship between ouabain binding and intracellular Na+ in patients receiving medication for hypertension (P less than 0.01) than in normotensive patients (P less than 0.05). These data suggest that patients receiving hypertensive medication may be less able to compensate than normotensive patients, (a) for the potential effect of Na+-K+-ATPase levels on intracellular Na+ and (b) for the potential effect of intracellular Na+ concentration on diastolic blood pressure. We propose that obese people with low levels of ouabain binding (primary obesity) may have an increased risk of developing hypertension if their compensatory mechanisms fail.

The effect of diet on ouabain binding to erythrocytes from obese subjects.

Ouabain binding to erythrocyte membranes is increased in obese subjects. Three study groups are compared: 14 reference subjects, 102 +/- 16% of ideal weight; 9 obese on unrestricted diets, 207 +/- 16% of ideal weight; 11 obese on restricted diets, 202 +/- 35% of ideal weight. A reproducible (CV = 11.3%) ouabain-binding assay is used to measure Na+-K+ ATPase sites in erythrocyte membranes. The number of binding sites per red blood cell for obese subjects on unrestricted diets, 431 +/- 30, is greater than for the reference group, 346 +/- 66 (p less than 0.01), or for obese subjects on restricted diets, 371 +/- 68 (p less than 0.05). These data suggest that caloric intake influences the number of Na+-K+ ATPase sites. Scatchard plots indicate only one type of binding site for ouabain with an affinity constant of about 3 X 10(8) M-1.

Improvement of the parallel flow dialysis technique for the detection of drug-binding proteins in column effluents.

The parallel flow dialysis technique was improved for application to the detection of drug-binding proteins in a column chromatographic effluent. To prevent the baseline drift, the pressures of both protein and drug channels were maintained equal during chromatography, and Brij-35 was added to the solvents. The improved method was successfully applied to the detection of methyl orange-binding proteins in human serum and bromphenol blue-binding proteins in rat liver homogenate.