Total Receptor Content Research Articles

The insulin resistant subphenotype of polycystic ovary syndrome: clinical parameters and pathogenesis

Objective This study was undertaken to compare clinical and biochemical characteristics of the insulin resistant (IR) and non-IR subphenotypes of polycystic ovary syndrome (PCOS). Study design Infertile PCOS women were classified as IR (n = 32) or non-IR (n = 46) on the basis of fasting glucose and insulin levels. The incidence of acanthosis nigricans (AN), hirsutism, and ovulation in response to clomiphene citrate (CC) was compared between the 2 groups, along with serum levels of gonadotropins, and sex steroids. Blood samples from 28 PCOS patients and 8 controls were analyzed by enzymatic immunoassay for autophosphorylated insulin receptor (APIR) and total insulin receptor (TIR) content. Results Insulin resistance was associated with obesity (odds ratio [OR] = 3.5, P < .05), AN (OR = 6.0, P < .05), hirsutism (OR = 3.1, P < .05), and resistance to CC (OR = 5.0, P < .05). Mean levels of LH, LH/FSH ratios, and testosterone were lower in women with IR (11.5 ± 6.8 mIU/mL, 2.0 ± 1.0, and 56.6 ± 29.0 ng/dL, respectively) compared with women without IR (15.0 ± 13.4 mIU/mL, 2.4 ± 1.5, and 72.5 ± 29.8 ng/dL, respectively) ( P < .05). Mean APIR/TIR ratios in IR women were lower than in non-IR women ( P < .05 at 100 nmol/L of insulin) and controls ( P < .01 at 1, 10 and 100 nmol/L insulin). Conclusion Patients with IR are more likely to be obese and have AN, hirsutism, resistance to CC, and lower LH, LH/FSH ratios, and testosterone levels. Furthermore, IR patients appear to have defective autophosphorylation of the insulin receptor, a key element in insulin action, and a possible mechanism for IR in PCOS.

Free-to-total leptin ratio in maternal plasma is constant throughout human pregnancy.

To clarify the mechanism of leptin resistance during pregnancy, we measured plasma leptin concentrations, free to total leptin ratio (percent free leptin) and soluble leptin receptor concentrations in pregnant women, and compared the results with those in non-pregnant women. We collected plasma samples from 23 non-pregnant and 31 pregnant women in the third trimester. Plasma samples from 5 pregnant women were collected longitudinally in each trimester. Plasma leptin concentrations in pregnant women in the second trimester (17.4 +/- 3.2 ng/ml) were higher than those in the first trimester of pregnancy (11.0 +/- 2.8 ng/ml, n = 5), as previously reported. However, percent free leptin did not change significantly throughout pregnancy. Percent free leptin correlated with total leptin concentrations (ng/ml) in non-pregnant women (r = 0.727, P < 0.0001), but not in women in the third trimester of pregnancy (r = 0.006). Constant percent free leptin during pregnancy despite increased leptin concentrations indicates increased leptin binding capacity in pregnant women, that might partly contribute to the establishment of leptin resistance. On the other hand, soluble leptin receptor concentrations showed significant negative correlation with BMI and plasma leptin concentrations in pregnant women (r = -0.470, P < 0.01 and r = -0.493, P < 0.01, respectively) but not in non-pregnant women. These data suggest the possibility that soluble leptin receptor is a minor component of leptin binding capacity in the plasma of pregnant women.

Properties of free and occupied androgen receptor in rat skeletal muscle cytosol: effect of testosterone

Our aim was to investigate the effect of a single testosterone (T) injection on the androgen receptor (AR) in rat skeletal muscle (SM) cytosol. The properties of AR were studied in order to establish the protocol for differential determination of free and hormone-occupied AR in SM cytosols from non-hormone-deficient animals. Using the developed ligand-exchange protocol, we demonstrated that injection of T (1 mg/kg) caused alternating changes of the total AR binding. The binding minimum (23% of the control) was measured 1 h after the injection. It was followed by pronounced and lasting elevation of the AR binding. In the control cytosols, AR complexes constituted ∼25% of the total receptor content. Changes of their relative content immediately after T administration were consistent with rapid nuclear translocation of the AR. Inhibition of protein synthesis by cycloheximide (CHI) injection demonstrated that delayed and lasting increase of the AR binding after T injection partially depended on the stimulated protein synthesis. Altogether, the obtained evidence supports the assumption that the AR mediates elevation of its own gene expression in SM upon administration of T.

Input-dependent synaptic targeting of alpha(2)-subunit-containing GABA(A) receptors in synapses of hippocampal pyramidal cells of the rat.

Pyramidal cells, expressing at least 14 subunits of the heteropentameric GABA(A) receptor, receive GABAergic input on their soma and proximal dendrites from basket cells, activating GABA(A) receptors and containing either parvalbumin or cholecystokinin and vasoactive intestinal polypeptide. The properties of GABA(A) receptors are determined by the subunit composition, and synaptic receptor content governs the effect of the presynaptic neuron. Using a quantitative electron microscopic immunogold technique, we tested whether the synapses formed by the two types of basket cell show a difference in the subunit composition of GABA(A) receptors. Terminals of one of the basket cells were identified by antibodies to parvalbumin. Synapses made by parvalbumin-negative terminals showed five times more immunoreactivity for the alpha(2) subunit than synapses made by parvalbumin-positive basket cells, whose synapses were frequently immunonegative. This difference is likely to be due to specific GABA(A) receptor alpha subunit composition, because neither synaptic size nor immunoreactivity for the beta(2/3) subunits, indicating total receptor content, was different in these two synapse populations. Synapses established by axo-axonic cells on axon initial segments showed an intermediate number of immunoparticles for the alpha(2) subunit compared to those made by basket cells but, due to their smaller size, the density of the alpha(2) subunit immunoreactivity was higher in synapses on the axon. Because the two basket cell types innervate the same domain of the pyramidal cell, the results indicate that pyramidal cells have mechanisms to target GABA(A) receptors, under presynaptic influence, preferentially to distinct synapses. The two basket cell types act via partially distinct GABA(A) receptor populations.

N-linked glycosylation is required for optimal AT1a angiotensin receptor expression in COS-7 cells.

The nature and role of glycosylation in AT1 angiotensin receptor (AT1-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT1a-Rs (HA-AT1a-Rs) in COS-7 cells. All three asparagine residues (Asn4, Asn176, Asn188) contained within consensus sites for N-linked glycosylation could be glycosylated in Cos-7 cells and appeared to be glycosylated on the endogenous AT1-R in bovine adrenal glomerulosa cells. Heterogeneity of glycosylation at each site accounted for the broad migration pattern of the AT1-R in SDS-PAGE. Mutation at each glycosylation site, either alone or in combination, had little effect on ligand binding parameters (although the N4K mutant had higher affinity) or signaling activity. However, an increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decreased surface expression of mutant HA-AT1a-Rs was correlated with decreased total cell receptor content as revealed by immunoblotting with an anti-HA antibody. These findings suggest that glycosylation enhances receptor stability, possibly by protecting nascent receptors from proteolytic degradation.

Some new insights into the effects of opioids in phasic and tonic nociceptive tests

In this review we critically appraise the value of some phasic and tonic nociceptive tests as models for differentiating the antinociceptive effects of opioid agonists. Using heat-evoked withdrawal of the hind paw or tail of a rodent, several early studies have assessed the effects of stimulus intensity upon antinociceptive potency of opioid agonists. After intrathecal (i.t.) administration of either morphine or sufentanil, for example, for any incremental change in stimulus intensity, the degree of right shift in the dose-response relationship was greater for morphine than for sufentanil. At first glance, such data appear to provide robust support for the pharmacological model of fractional receptor occupancy (FRO), which, according to the historical tenets of classical receptor theory, describes the relationship between intrinsic efficacy and the total receptor concentration. However, new data which elegantly characterize the relative contribution of small calibre unmyelinated and myelinated nociceptive afferents in mediating thermal-evoked responses challenge the exclusivity of such explanations with origins in classical theory. Within our review we report the results of experiments which provide direct electrophysiological evidence that noxious skin heating at a low rate activates C-polymodal nociceptors, but does not effectively activate A-delta mechanothermal nociceptors. In contrast, a high rate of skin heating activates both nociceptor classes, but produces a more intense activation of A-delta nociceptors that occurs after a shorter onset latency compared with the activation of C-fibre nociceptors. Thus, in direct challenge to the traditional model of FRO, a shift in the dose-response relationship of morphine to the right with a reduction in efficacy, may reflect the limited effectiveness of morphine to attenuate the A-delta-mediated component which assumes increasing dominance at high intensity heating. In our appraisal of other nociceptive models we provide an in-depth characterization of afferent processing in the early neonate rat, in which opioids have been tested in both phasic (tail flick and hot-plate) and tonic (formalin) tests. Afferent processing in this model is typified by several behavioural, anatomical and functional features which, although not pathological, are characteristic of those observed in models of nerve injury using the adult rat. Notably, these features include a lack of segmental inhibition (`disinhibition') and afferent input in large diameter myelinated fibres which make synaptic contacts within superficial laminae of the dorsal horn that in the adult are predominantly nociceptive. Paradoxically, because this paradigm demonstrates increased sensitivity to the antinociceptive effects of opioids it may have special merit as a model of tonic pain. It was recently announced that the i.t. administration of pertussis toxin (PTX) caused hyperalgesia and allodynia that appears similar to the symptoms reported by patients suffering from neuropathic pain. Unlike the effects of other opioids so far tested, buprenorphine-induced antinociception is not blocked in this model. This is an exciting finding and provides new optimism that some opioids, notably buprenorphine, may have a special role in managing some types of neuropathic pain.

Stimulation of 125I-transferrin binding and 59Fe uptake in rat adipocytes by vanadate: Treatment time determines apparent tissue sensitivity

Vanadium compounds have been documented to stimulate a number of insulin biological effects in vitro and in vivo. We previously demonstrated stimulation of glucose transport and insulin-like growth factor-II (IGF-II) binding in rat adipocytes. These actions are associated with translocation of glucose transporters and IGF-II receptors from an intracellular compartment to the plasma membrane. The transferrin receptor is also recruited to the plasma membrane in response to insulin. Freshly isolated rat adipocytes were incubated with vanadate and insulin at 37°C, and after treating the cells with KCN to inhibit further receptor movement, diferric 125I-transferrin binding was assayed. Vanadate stimulated a dose- and time-dependent increase in 125I-transferrin binding, reaching maximum (∼threefold) stimulation at 1 mmol/L after a 4-hour incubation. This was equivalent to the maximum insulin effect that was obtained with 10 −8 mol/L after 30 minutes. A similar degree of stimulation was achieved with 0.1 mmol/L vanadate after 8 hours of exposure. Dose-response data showed that the apparent sensitivity to vanadate was time-dependent and increased with the duration of exposure (EC 50: 30 minutes, 1 mmol/L; 3 hours, 0.35 mmol/L). Scatchard analysis of 125I-transferrin binding showed that both insulin and vanadate increased receptor binding capacity with no effect on receptor affinity. Total cellular transferrin receptor content measured by immunoblotting with monoclonal anti—transferrin receptor antibody (OX-26) was not altered by insulin or vanadate, consistent with receptor translocation. Assessment of 59Fe uptake from 59Fe-labeled diferric transferrin showed that vanadate augmented 59Fe uptake in a dose-dependent manner to an extent similar to insulin, demonstrating the functional activity of the receptors (percent of control: 10 −8 mol/L insulin, 175% ± 23.8%, P < .02; 0.3 mmol/L vanadate, 188% ± 17.3%, P < .01). We conclude that vanadate mimics insulin to augment cell surface transferrin receptors and increase Fe uptake in rat adipocytes. The time-dependent apparent increase in sensitivity is consistent with the effectiveness of very low concentrations of vanadate in vivo after several days of administration, and suggests a requirement for vanadate entry into cells to mediate this biological response.

Circulating Transferrin Receptor Assay—Coming of Age

Transferrin receptors are transmembrane proteins present on the surface of most cells. Under normal circumstances, the iron required for cellular metabolism is acquired via transferrin receptors. Several recent reviews detail the methods of iron acquisition, the intracellular throughput of transferrin receptors, and the controlling mechanisms in the cell‘s quest to acquire and store iron (1)(2)(3). The cells of different organ systems show considerable differences in the concentration of cellular transferrin receptor, the highest concentrations being found in cells of organs with the highest iron requirements, such as the erythroid bone marrow and placenta (4). The concentration of cell surface transferrin receptor is carefully regulated by transferrin receptor mRNA according to the internal iron content of the cell and its individual iron requirements. Iron-deficient cells contain increased numbers of receptor, while receptor numbers are downregulated in iron-replete cells (4)(5). Transferrin receptor was identified in serum (6) after investigators recognized that the molecule was secreted into culture media in reticulocyte and erythroleukemia cell models (7)(8), and the concentrations of secreted receptor were found to correlate with the total receptor content of the cells. Subsequent studies showed that serum concentrations of transferrin receptor increase in iron-deficiency anemia, making it a useful marker in the diagnosis of microcytic anemias (9). Circulating transferrin receptor is a truncated form of tissue receptor (10), produced by proteolytic cleavage of cellular receptor, and for the most part circulates attached to transferrin (11)(12). The reference interval for concentration of circulating receptor varies between different assay systems, depending on the choice of calibrators. In this issue of Clinical Chemistry , the transferrin receptor assay recently approved by the US Food and Drug Administration is described by Allen et al. (13). The calibrators used in this enzyme immunoassay system are …

Effects of Chinese Kidney-Tonifying herbal medicine on M-cholinergic receptor and acetylcholinesterase activity in dementia mimetic mice

Objective: In order to study the effects of Chinese Kidney-tonifying (KT) herbal medicine on the M-cholinergic receptor(M-R) and the acetylcholinesterase(AchE) activity in the models’ cerebral cortex.Methods: Dementia mimetic mice models were formed with aluminum chloride.Results: The Muscarine receptor total receptor concentration (M-Rt) lowered and the AchE increased in the model significantly compared with healthy young mice. KT could markedly reduce the increase of AchE and elevate the lowered M-Rt in cerebral cortex of the dementia mimetic mice and improve the memory.Conclusions: KT is effective in preventing the degeneration of cerebral function and presenile dementia.

Epidermal growth factor receptors in rat ovarian tissue

Epidermal growth factor is known to have mitogenic effects in the ovary. To determine whether this effect is receptor-mediated, and whether the receptor number changes during the estrus cycle, in this study we have evaluated the total and occupied EGF receptor (EGFr) concentrations and receptor binding characteristics in adult rat ovaries collected at diestrus and early estrus. These represent stages before and after the pituitary LH/FSH surges of the estrus cycle. The 100 000 g plasma membrane fractions were isolated and EGFr concentrations evaluated with and without 1 mM EDTA treatment at pH 4.5 in order to release receptor-bound ligand and therefore assess the unoccupied and total receptor concentrations. EGF receptor affinity and number were analyzed by Scatchard analysis. No statistically significant difference between unoccupied and total receptor concentration in the diestrus phase ovaries was found. The percent-age of occupied receptors (total-unoccupied) in diestrus membranes was 7.1%. In early estrus ovaries, a significant increase ( P < 0.05) was observed in total receptor concentration when compared with the number of unoccupied receptors. The percentage of occupied receptors in early estrus membranes was 35.7%. Comparison between the ovaries from the two phases indicated an almost twofold increase in total EGF receptor number in early estrus membranes when compared with diestrus membranes. Furthermore, the percentage of occupancy between the two studied groups indicate a significant increase ( P < 0.05) in the number of occupied receptors in early estrus when compared to diestrus. Thus, we have demonstrated that not only EGF receptor concentrations are modulated through the rat estrus cycle, but also the concentration of EGF-like substances bound to such receptors.

Autoradiographic evidence that (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB) displays in vivo selectivity for the muscarinic m2 subtype

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorinated derivative of QNB, (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[ 125I]IQNB) binding by unlabelled (R,S)-FMeQNB. In the absence of (R,S)-FMeQNB, (R,S)-[ 125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 75 nmol of (R,S)-FMeQNB, (R,S)-[ 125I]IQNB labelling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that (R,S)-FMeQNB is m2-selective in vivo, and that (R,S)-[ 18F] FMeQNB may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Ontogenesis of LH and FSH receptors in postnatal rabbit testes: age-dependent differential expression of long and short RNA transcripts.

The ontogeny of testicular LH and FSH receptors was studied in New Zealand rabbits from 20 to 180 days postpartum. The concentrations of free receptors (per mg total proteins) were very low at day 20. They increased steeply at day 30 for the LH receptor and at day 50 for the FSH receptor. Three RNA bands (1.2, 2.5 and 3 kb) were repeatedly detected on northern blots for the LH receptor and two bands (1.2 and 2.2 kb) were detected for the FSH receptor. The 1.2 kb band (which cannot give rise to full-length, membrane-anchored receptor) was present throughout the 20-180 day period for each receptor. However, the higher molecular mass bands were nearly undetectable at day 20. The 2.5 and 3 kb bands of the LH receptor increased twofold between day 20 and day 120, while the 2.2 kb band of the FSH receptor increased fivefold between day 20 and day 75. Thus the very low concentrations, or even absence, of the larger transcripts of both LH and FSH receptors were correlated with the inability to detect their cognate protein until 20 days of age. Subsequently, coordinated increases in high molecular mass transcripts and protein were observed for both receptors. Total LH receptor content increased in parallel to the previously reported increase in plasma testosterone between day 65 and day 100. FSH receptor density began to increase steeply at day 50, just at the onset of spermatogenesis. Thus, postnatal testicular development in the rabbit seems to entail the transcription of high molecular mass, translatable transcripts of the gonadotrophin receptors.

Autoradiographic Evidence That 3-Quinuclidinyl-4-fluorobenzilate (FQNB) Displaysin VivoSelectivity for the m2 Subtype

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood–brain barrier. We now demonstrate thein vivom2 selectivity of a fluorine derivative of QNB (FQNB), by studying autoradiographically thein vivoinhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled FQNB. In the absence of FQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 30.0 nmol of racemic FQNB, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that FQNB is m2-selectivein vivoand that [18F]FQNB or a closely related analogue may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD.

Regulation of estrogen receptor concentrations in the rat brain: effects of sustained androgen and estrogen exposure.

To determine whether estrogen and androgens either alone or in combination downregulate estrogen receptors in the brain, ovariectomized/adrenalectomized female rats received one of the following four treatments: (1) one subcutaneously placed Silastic capsule containing 10% estradiol in cholesterol, (2) one capsule containing 10% estradiol and two capsules containing 100% 5 alpha-dihydrotestosterone (DHT), (3) two capsules containing DHT, or (4) empty Silastic capsules (control animals). Animals were killed 4 or 8 days after capsule insertion and the occupied, unoccupied and total estrogen receptor content in specific brain nuclei was determined by quantitative in vitro autoradiography. To determine if the effects of the androgen were reversible, DHT capsules were removed after 4 days from half of the estradiol+DHT-treated rats, and the animals were killed 4 days later. Estradiol downregulated estrogen receptor expression in the periventricular preoptic area, medial preoptic area, bed nucleus of the stria terminalis (BNST), arcuate nucleus (ARC), ventromedial nucleus (VMN), and medial and cortical amygdala, decreasing receptor content by 30-41% in animals treated for 4 days, and by 44-60% in animals treated for 8 days with estradiol alone. DHT treatment in combination with estradiol further decreased estrogen receptor content in the BNST, ARC and VMN, relative to the estradiol-only animals. DHT in the absence of estrogen was without effect. In animals in which the DHT capsules were removed after 4 days of exposure, allowing the estradiol to remain for a further 4 days, estrogen receptor levels were indistinguishable from those measured in control animals treated for 8 days with estradiol alone. These results demonstrate that sustained estrogen exposure downregulates levels of estrogen receptor in the brain and confirm that DHT synergizes with estrogen in inducing this response in some, but not all, target neuronal groups.

Biochemical, metabolic and immune correlates of seasonal variation in violent suicide: a chronoepidemiologic study

The purpose of this chronoepidemiologic study was to investigate the time-relationships between the yearly variations in occurrence of violent suicide in Belgium and the yearly variations in various biochemical, metabolic and immune variables in the Belgian population. The weekly mean number of deaths due to violent suicide for all of Belgium for the period 1979-1987 was computed. Twenty-six normal volunteers had monthly blood samplings during one calendar year for assays of plasma L-tryptophan (L-TRP), competing amino acids (CAA), and melatonin levels, maximal [3H]paroxetine binding to platelets, serum total cholesterol, calcium, magnesium, and soluble interleukin-2 receptor concentrations, and number of CD4+ T, CD8+ T and CD20+ B lymphocytes. The annual rhythm in violent suicide rate is highly significantly synchronized with the annual rhythms in L-TRP, [3H]paroxetine binding, cholesterol, calcium, magnesium, CD20+ B cells, and CD4+/CD8+ ratio; the mean peak (violent suicide, [3H]paroxetine binding) or nadir (all other variables) occurs around 3 May. There were significant inverse time-relationships between the time series of violent suicide rate and L-TRP, L-TRP/CAA ratio, total cholesterol, calcium and magnesium, CD4+/CD8+ T cell ratio and number of CD20+ B cells. Maximal [3H]paroxetine binding to platelets was significantly and positively related to the time series of violent suicide. An important part (56.4%) of the variance in mean weekly number of violent suicide rate was explained by the time series of L-TRP, cholesterol and melatonin.

A novel cis-acting element in a liver cytochrome P450 3A gene confers paradoxical, synergistic induction by glucocorticoids plus antiglucocorticoids : [formula omitted]†[formula omitted]∗ Department of Medicine, Hepatobiliary Research Center, Section of Medical Toxicology, University of Colorado Health Sciences Center∗ and Department of Pathology, Medical College of Virginia †

The specific binding of the archetypal cytochrome P450 3A subfamily (CYP3A) inducer dexamethasone is examined in microsomal fractions since rat and human liver CYP3A is induced by glucocorticoids through a mechanism which is apparently independent of the cytosolic glucocorticoid receptor. Dexamethasone binds in a specific and saturable manner to microsomes with an affinity constant (Kd) of 59 ± 12.9 nM which compares to a Kd of 2.3 ± 0.17 nM in cytosol. The total receptor concentrations ([LR]maxe) in microsomes and cytosol are 9.5 ± 1.67 pmols/mg protein and 410 ± 167 fmol/mg protein respectively. The microsomal binding of dexamethasone is antagonised by several transcriptional and/or post-transcriptional CYP3A inducers with decreasing potency pregnenolone 16α carbonitrile > metyrapone > phenobarbitone. Troleandomycin, which indirectly induces CYP3A1 in vivo and by protein stabilisation, does not antagonise the binding of dexamethasone in microsomes. The transcriptional and/or posttranscriptional induction of CYP3A may therefore be associated with the interaction of inducers with a microsomal protein.

Autoradiographic Evidence That Quinuclidinyl 4-(Bromophenyl)-2-thienylglycolate (QBPTG) Displays in Vivo Selectivity for the Muscarinic m2 Subtype

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of an analogue of QNB, 4-(bromophenyl)-2-thienylglycolate (QBPTG), by studying autoradiographically the in vivo inhibition of radioiodinated ( R)-3-quinuclidinyl ( S)-4-iodobenzilate (( R,S)-[ 125I]IQNB) binding by unlabeled QBPTG in rat brain. In the absence of QBPTG, ( R,S)-[ 125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 37.5 nmol of racemic QBPTG, ( R,S)-[ 125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that QBPTG is m2-selective in vivo and that [ 76Br]QBPTG, or a radiofluorinated analogue, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD. In addition, a radioiodinated analogue may be of potential use in single photon emission tomographic studies.

Ageing and prostate: age-related changes in androgen receptors of epithelial cells from benign hypertrophic glands compared with cancer

Total and nuclear androgen receptors (AR) were studied from epithelial cells in internal and external prostatic zones in 51- to 86-year-old individuals with benign prostatic hyperplasia (BPH) ( n = 68) and prostatic cancer ( n = 9). We focussed on the role played by androgens on those processes, despite the fact that at these ages, its secretion has normally decreased. In BPH, the nuclear AR do not change, but total measured androgen receptors rise with age ( r = 0.5, P < 0.01). Total or nuclear AR do not correlate with gland volume, despite its increase with age ( r = 0.8, P < 0.05). In prostates less than 180 cc in volume, there is a significant correlation between size, serum total testosterone level ( r = 0.53, P < 0.05) and prostatic specific antigen (PSA) ( r = 0.63, P < 0.05). The amount of nuclear AR in cells from the external zone (infiltrated by cancer or healthy) is two times greater than in those from the internal region. Total receptor content of the external zone cells is also high, but the sample is too small to demonstrate an age dependence. The results suggest that ageing is accompanied by an accumulation of non-nuclear AR in the cytosol, that does not play a role in the development of BPH because the amount of nuclear receptors remains unaltered. The enrichment in nuclear receptors of the external zone cells, independently of the presence of cancer, points to a greater androgen dependence in these cells than in cells of the internal region.

A snake toxin against muscarinic acetylcholine receptors: Amino acid sequence, subtype specificity and effect on guinea-pig ileum

The sequence of muscarinic toxin 1 (MT1) from Dendroaspis angusticeps (green mamba) was determined (66 amino acids, M r 7509). The central part, peptide 25–40, is rich in hydrophobic amino acids, which is a characteristic of muscarinic toxins. MT1 started to inhibit [ 3H]-NMS ( N-methylscopolamine) binding to synaptosomal membranes of porcine brain (contains all five receptor subtypes) at about 1 nM and to membranes from pig heart muscle (only subtype m2) at about 1 μM. Binding of [ 3H]-AF-DX 384 to heart was inhibited with an ic 50 of 14 μM and to brain in two steps. In the first step (IC 50 = 32 nM) binding decreased by 37%, indicating that the toxin acted on m1 or m4 receptors, each accounting for about 40% of total receptor content. The second step was similar to the effect on heart. Pirenzepine inhibited binding of [ 125I]-MT1 to brain receptors with an ic 50 of 6.5 nM, corresponding to a K i, of about 6 nM. Literature values of K i for pirenzepine are 16–18 nM for m1 and ⩾ 120 mM for other subtypes. This indicates binding to m1 receptors. [ 125I]-MT1 bound to brain with a K d of 20 nM and a Hill coefficient of 1.0 i.e. one toxin molecule per receptor. In guinea-pig ileum, MT1 (670 nM) produced a rapid contraction, reversible by atropine. The toxin may be an agonist, but might also cause contraction by inducing acetylcholine release by a different mechanism.

Autoradiographic Evidence That QNB Displays in Vivo Selectivity for the m2 Subtype

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We have previously reported the results of in vivo dissection studies, using both carrier-free and low specific activity [ 3H]QNB, which show that [ 3H]QNB exhibits a substantial in vivo m2 selectivity. Because of the expense of the radioligand and the long exposure time required for the X-ray film, performing a large number of direct in vivo autoradiographic studies using [ 3H]QNB is precluded. Therefore, we now confirm these results autoradiographically by studying the in vivo inhibition of radio-iodinated ( R)-3-quinuclidinyl ( S)-4-iodobenzilate (( R,S)-[ 125I]IQNB) binding by unlabeled QNB. In the absence of QNB, ( R,S)-[ 125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 15 nmol QNB, ( R,S,)-[ 125I]IQNB labeling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that QNB is m2-selective in vivo and that a suitably radiolabeled derivative of QNB, possibly labeled with 18F, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD.