Diurnal variation in insulin binding to human monocytes.

We studied insulin binding to monocytes isolated from dieting and fasting young healthy persons during a 24-h period. In the persons who ate their usual food, we found a characteristic diurnal variation in the cellular insulin binding. Daytime insulin binding was low with a minimum in the afternoon; binding increased during the evening, reaching a maximum in the early morning. This variation seemed to be diet related, as total fasting abolished the circadian rhythm. Changes in cellular insulin binding were due to fluctuations in the binding affinity rather than alterations in receptor concentration. The circadian variation in insulin binding was grossly parallel to the well known variations in insulin sensitivity and glucose tolerance during the day.     

Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus

We used quantitative in vitro autoradiography to localize and characterize 2-125I-melatonin binding sites in the rat suprachiasmatic nuclei in relation to pineal melatonin production. In a light:dark cycle of 12:12 h, binding density exhibited significant diurnal variation with a peak at the dark-light transition and a trough 12 hours later. Saturation studies suggested that the decreased binding at light-dark transition might be due to a shift of the putative melatonin receptor to a low affinity state.     

The role of the adrenal in generating the diurnal variation in circulating levels of corticosteroid-binding globulin in the rat

Corticosteroid-binding globulin (CBG) levels were measured in serum samples collected sequentially from rats into which indwelling catheters had been inserted. A distinct diurnal variation in CBG levels was found, with the highest levels of binding protein at the beginning of the dark period. CBG levels then decreased until a nadir was reached shortly after the beginning of the light period. To examine the role of glucocorticoid in the generation of this pattern, rats were adrenalectomized 10 days before repeating the experiment. Adrenalectomy abolished both the diurnal variation in CBG levels and the high degree of variation in the levels of binding protein between animals. However, adrenalectomy just before the onset of the dark period did not eliminate the expected decrease in CBG levels. To further explore the role of steroid, animals that had been adrenalectomized for 10 days were given saline containing 25 micrograms/ml corticosterone for a period of 24 h. Despite the attainment of normal plasma corticosterone levels, no decrease in CBG levels was observed. When steroid administration was discontinued, however, CBG levels dropped concurrently with the decreasing steroid. These studies show that a diurnal variation in CBG exists and suggest that it is the result of the diurnal variation in glucocorticoid levels.     

Rhythmic expression of functional MT1 melatonin receptors in the rat adrenal gland

We previously demonstrated that melatonin is involved in the regulation of adrenal glucocorticoid production in diurnal primates through activation of MT1 membrane-bound melatonin receptors. However, whether melatonin has a similar role in nocturnal rodents remains unclear. Using an integrative approach, here we show that the adult rat adrenal gland expresses a functional MT1 melatonin receptor in a rhythmic fashion. We found that: 1) expression of the cognate mRNA encoding for the MT1 membrane-bound melatonin receptor, displaying higher levels in the day/night transition (1800-2200 h); 2) expression of the predicted 37-kDa MT1 polypeptide in immunoblots from adrenals collected at 2200 h but not 1000 h; 3) no expression of the MT2 melatonin receptor mRNA and protein; 4) specific high-affinity 2-[(125)I]iodomelatonin binding in membrane fractions and frozen sections from adrenals collected at 2200 h but not 0800 h (dissociation constant = 14.22 +/- 1.23 pm; maximal binding capacity = 0.88 +/- 0.02 fmol/mg protein); and 5) in vitro clock time-dependent inhibition of ACTH-stimulated corticosterone production by 1-100 nm melatonin, which was reversed by 1 microm luzindole (a melatonin membrane receptor antagonist). Our findings indicate not only expression but also high amplitude diurnal variation of functional MT1 melatonin receptors in the rat adrenal gland. It is conceivable that plasma melatonin may play a role to fine-tune corticosterone production in nocturnal rodents, probably contributing to the down slope of the corticosterone rhythm.     

Diurnal variation in FEV1 after heart-lung transplantation.

We have examined the diurnal variation in forced expiratory volume in one second (FEV1) in 25 heart-lung transplantation patients over a four week period in order to study the pathophysiological mechanisms underlying the increased mortality and morbidity which occurs at night in asthma. These patients do not have pulmonary autonomic nervous reflexes, but often have muscarinic receptor hypersensitivity. They also develop mixed cell infiltration of the lung tissue in the course of infection or rejection. Thus, they show many features in common with asthma. Seventeen patients (68%) showed a significant diurnal variation in airway calibre (mean amplitude of FEV1 was 4.6% (SD 3.7%)), which is similar to that reported in normal adults. One patient had a diurnal variation of 34.5% during an episode of rejection. This variation fell to 6.9% after steroid therapy, a change often seen in asthma. There was a correlation between increased amplitude of the variation and the presence in transbronchial biopsies of airway submucosal eosinophils and lymphocytes, associated with a histological diagnosis of acute rejection and with epithelial damage. No association was seen with muscarinic receptor sensitivity. The variation in FEV1 showed no alteration from the normal day/night synchronization, and the peak values were around 1300 h. We conclude that the diurnal variation in FEV1 after heart-lung transplantation is not dependent on autonomic nerve reflexes or muscarinic receptor sensitivity, but is related to the consequences of inflammation described above.     

Decreases in mediobasal hypothalamic and preoptic area opioid ([3H]naloxone) binding are associated with the progesterone-induced luteinizing hormone surge

In view of evidence implicating hypothalamic opioid systems in the control of LH release, we have examined the binding of [3H]naloxone (NAL) to slices of mediobasal hypothalamus (MBH) and preoptic area (POA) during the induction of an afternoon LH surge (1630-1700 h) in estradiol benzoate (EB)-primed ovariectomized (OVX; day 0) rats by treatment with progesterone (P; day 2). Such a surge was invariably accompanied by a decrease from early morning (1000 h) values in the number of NAL-binding sites detectable in the MBH, while the affinity of the binding site was not affected over the course of the day. Time-course studies indicated that P injection at 1000 h on day 2 was followed by a transient midday elevation in the amount of NAL bound to slices of MBH; the binding decreased significantly before the onset of and during the LH surge. A similar diurnal change was not observed in MBH slices of either oil-treated OVX rats (controls) or EB-treated OVX rats, which displayed only a 2-fold increase in LH release in the afternoon. Further studies indicated a similar change in NAL binding to slices of the POA of EBP-treated rats. Since hypothalamic opioid systems inhibit LH release, the decrease in opioid binding to MBH as well as POA slices suggests that P may curtail the existing opioid inhibitory influence in these areas before and during the course of the afternoon LH surge.     

Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain

In the rat brain, the binding of corticosterone is mediated through two receptor types, the type I receptor and the type II receptor, which are presumed to be encoded by genes designated as MR and GR, respectively. We have studied the regulation of these receptors by glucocorticoids, utilizing a cytosol receptor binding assay. In addition, we have employed molecular probes for the GR and the MR to measure receptor mRNAs. The level of type II receptor binding is uniform across several brain regions, as is the expression of GR (type II) mRNA. In contrast, type I receptor binding is concentrated in the hippocampus, and the MR (type I) mRNA similarly shows a higher level of expression in hippocampus than in the other brain regions studied. Removal of endogenous glucocorticoids by adrenalectomy (ADX) induces an increase, and corticosterone administration results in a decrease, in the level of type I and type II binding in the hippocampus; however, no significant changes in the MR (type I) or GR (type II) mRNA levels are seen with these treatments. The diurnal variation of serum corticosterone in intact rats is correlated with a circadian regulation of type I receptor binding in the hippocampus, while MR (type I) mRNA expression is unaffected. Thus, the changes in type I and type II receptor binding capacity elicited by differing steroid conditions cannot be attributed to modulation of the steady state levels of MR (type I) or GR (type II) mRNA.     

Quantitative autoradiographic delineation of the distribution of beta-adrenergic receptors in canine and feline left ventricular myocardium

The distribution of adrenergic receptors in specific components of the heart such as vessels and myocytes cannot be determined easily with assays of membranes prepared from homogenates of whole tissue. Accordingly, we characterized the binding of the potent nonsubtype selective antagonist [125iodo]cyanopindolol to beta-receptors in unfixed transmural slices of feline and canine left ventricle. Specific binding ratios greater than 90% were achieved at radioligand concentrations near Kd and greater than 80% at saturating ligand concentrations. Binding of radioligand to receptors in transmural slices was rapid, saturable, stereoselective, and displaceable by antagonists and agonists with the rank order of potency expected of beta-adrenergic receptors. Analysis of binding isotherms indicated maximum binding capacities of 27.8 +/- 6.6 and 40.6 +/- 5.1 fmol/mg tissue protein and dissociation constants of 10.1 +/- 1.8 and 21.3 +/- 1.6 pM in feline and canine ventricular slices, respectively. The distribution of beta-receptors in myocytes and selected vascular components of the heart was determined with quantitative film autoradiography and high resolution computer-based analysis and display of the density of binding sites, maximum binding capacity, and binding affinity measurements. The results of autoradiographic analysis revealed a uniform transmural distribution of receptors in regions composed primarily of ventricular myocytes but an inverse relation between the density of beta-receptors and the diameter of coronary vessels. Large epicardial conductance arteries had half the receptor density of subjacent myocytes; small mural arteries had approximately 60% of the beta-receptor density of nearby myocytes, and the coronary resistance arterioles had the highest receptor density of any vascular compartment, which was equivalent to that of myocytes. The methods developed should be of particular value in characterizing the distribution and function of receptor subtypes and mechanisms of regulation of adrenergic responsiveness in intact myocardium.     

beta-Adrenergic receptor regulation by agonists and membrane depolarization in rat brain slices.

Rat cerebral cortex slices exposed to (-)-isoproterenol and then washed accumulated significantly less cyclic AMP when rechallenged with isoproterenol than did control slices. The isoproterenol-induced desensitization was associated with a concurrent reduction in [3H]dihydroalprenolol membrane binding but with no change in the affinity of [3H]dihydroalprenolol or isoproterenol for the binding sites. beta-Adrenergic receptor desensitization was rapidly reversed by slice depolarization with high-[K+] buffers, batrachotoxin, grayanotoxin, or veratridine, even in the continued presence of isoproterenol. Restoration of binding by grayanotoxin was prevented by tetrodotoxin or by removing Na+ from the buffer. These data demonstrate partial participation of the membrane receptor in beta-adrenergic desensitization of rat brain slices and suggest that brain beta-adrenergic receptors, like cholinergic receptors in skeletal muscle and alpha-adrenergic receptors in rat parotid, are regulated in part by membrane voltage.     

Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: daily and seasonal variations

In postmetamorphic Xenopus laevis liver cytosol the glucocorticoid binding capacity R0 and the dissociation constant Kd were determined. The receptor assay included an incubation period of 24 hr at 0-4 degrees with sodium molybdate to stabilize the receptor. Dexamethasone, triamcinolone acetonide, and corticosterone as tritiated ligands were compared regarding the R0 (67.6, 57.2, and 30.7 fmol/mg protein), the Kd (3.54, 0.56, and 9.03 nM), and the rate of dissociation in young specimens of X. laevis. In adult toads the [3H]dexamethasone receptor binding capacity was threefold higher in females than in males (153.86 +/- 12.19, 54.29 +/- 4.5 fmol/mg protein)--with about the same Kd (3.97 +/- 0.57, 4.08 +/- 0.28 nM). Young toads were kept under an artificial light regime (light from 600 to 1800 hr) and dexamethasone binding was measured every 3 hr. Unlike Kd, R0 showed a significant diurnal variation with maximal values at 600 and 1800 hr, which occurred about 9 hr after a maximal level of corticosterone in serum was reached (900, 2100). Seasonal variations of the [3H]dexamethasone and [3H]corticosterone binding capacity were different in both sexes of adult X. laevis. Maximal values in males were found in June/July and October/November. In females, the R0 was increased in the second half of the year with the maximum in August (275.5 +/- 45.02 fmol/mg protein). No correlation between R0 and the concentrations of corticosterone or aldosterone existed.     

神经性厌食患者的单个核白细胞糖皮质激素受体及昼夜节律改变

应用放射配体结合法测定10例神经性厌食患者外周血单个核白细胞(MNL)的糖皮质激素受体(GR)。结果表明GR的最大结合容量(R_0)明显低于对照组,且早晚差异消失。GR的平衡解离常数(Kd)无明显改变。同时测定了血浆皮质醇(F)浓度,其浓度明显高于正常人,且周日节律也消失。提示病变环节包括下丘脑,同时较为满意的解释了神经性厌食患者血浆F增高,但无F增多症的临床表现的矛盾现象。     

Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.     

TR(beta)1 protein is preferentially expressed in the pericentral zone of rat liver and exhibits marked diurnal variation

We investigated the distribution and diurnal variation of TR(beta)1 protein expression in liver with specific antibodies against TR(beta)1. Immunohistochemistry showed a zonal distribution of TR(beta)1 with maximum expression in the pericentral zone matching some known T(3)-responsive enzyme activities in the liver, such as glutamine synthetase, cholesterol 7alpha- hydroxylase, and spot 14. Combining immunohistochemistry and image analysis we found and quantified the same zonal distribution for 5'-deiodinase type 1 as for TR(beta)1. Western blot analysis revealed a profound diurnal variation for TR(beta)1 protein expression, with highest levels at the beginning of the dark period. TR(beta)1 diurnal variation partly overlaps with the T(3)-responsive genes, cholesterol 7alpha-hydroxylase and spot 14. Furthermore, TR(beta)1 distribution along the porto-central axis does not change during the day, indicating that the zonal expression of TR(beta)1 is stable. This is the first time that zonal distribution in liver has been demonstrated for a member of the nuclear receptor family. This finding together with the observed diurnal rhythm has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.     

Mammary gland prolactin receptor and pituitary prolactin secretion in lactating mice with different lactational performance

SHN female mice, a high mammary tumour strain, are superior to SLN, a low mammary tumour strain, in lactational performance. Mammary gland prolactin receptor and pituitary prolactin secretion during lactation were compared between these strains. The binding activity, the number of receptor sites per mg tissue and the association constant were measured by the in vitro incubation of mammary gland slices with 125I-labelled bovine prolactin, and the pituitary and plasma levels of prolactin were assayed by homologous radioimmunoassay. There was only a slight difference between strains in any of the parameters for prolactin receptor and for prolactin secretion on either day 4 or day 9 of the first lactation. Almost all the correlation coefficients between each parameter for prolactin receptor and the pituitary or plasma level of prolactin were not statistically significant. These findings suggest that any parameter for prolactin examined in this study is not always directly indicative of lactational performance and further show that the individual variation in the pituitary prolactin secretion during lactation is not so great as to alter the prolactin receptor.     

Diurnal variation in growth hormone receptor messenger ribonucleic acid in liver and skeletal muscle of lit/+ and lit/lit mice

The present study investigated the diurnal variation in GH receptor (GHR) mRNA in liver and skeletal muscle of 3-month-old GH-deficient and -sufficient mice using quantitative real-time RT-PCR. lit/lit (GH deficient) or lit/+ (GH sufficient) mice were fed ad libitum and lights were on between 0600 and 2000. Tissues were collected at 0800-1000, 1200-1400 and 2000-2200. Hepatic GHR mRNA levels of lit/+ mice at 0800-1000 were significantly lower than those at 1200-1400 and 2000-2200. There was no significant variation in hepatic GHR mRNA of lit/lit mice. In skeletal muscle, GHR mRNA levels of both lit/+ and lit/lit mice at 1200-1400 were significantly higher than those at 0800-1000 and 2000-2200. There was also a diurnal change in hepatic IGF-I mRNA levels of lit/+ but not of lit/lit mice; the levels were lowest at 0800-1000 in lit/+ mice. On the other hand, there was no variation in IGF-I mRNA levels in skeletal muscle. These results suggest that 1) there is a diurnal variation in GHR expression in liver and skeletal muscle and the pattern of the variation is tissue specific; 2) GH deficiency blunted the diurnal variation in GHR mRNA in liver but not that in skeletal muscle; 3) IGF-I mRNA expression in liver is more closely related to GHR mRNA expression than that in skeletal muscle.     

Interconverting mu and delta forms of the opiate receptor in rat striatal patches.

The binding of a radiolabeled "mu receptor" prototype opiate, dihydromorphine (H2morphine), and the binding of a "delta receptor" prototype, [D-Ala2,D-Leu5]enkephalin (D-Enk), to slide-mounted rat caudate slices were simultaneously compared quantitatively and visualized by autoradiography. Generally, D-Enk bound to opiate receptors distributed evenly throughout the entire striatum (type 2 pattern), whereas H2morphine labeled discrete islands or patches of receptors (type 1 pattern). In the presence of Mn2+ (3 mM) or other divalent cations, however, Na+ and GTP at 25 degrees C caused an increase in D-Enk binding at the expense of H2morphine binding at striatal opiate receptor patches. Thus, these conditions shifted D-Enk binding from an even pattern to one that included both an even and patchy distribution. These incubation conditions not only promoted D-Enk binding to striatal patches but also enabled the opiate receptor to regulate adenylate cyclase with the same (P less than 0.01) ligand selectivity pattern as that obtained by the displacement of D-Enk binding. The relative affinity of opiate receptors in striatal patches for opiate peptides, naloxone, and morphine appears to be a function of incubation conditions and coupling to adenylate cyclase and is not indicative of distinctly different opiate receptors. We postulate a three-state allosteric model consisting of mu agonist-, mu antagonists-, and adenylate cyclase-coupled delta-agonist-preferring states, whose equilibrium may be regulated by a sulfhydryl group mechanism.     

Renal receptor-binding activity of reduced metabolites of aldosterone: evidence for a mineralocorticoid effect outside of the classic aldosterone receptor system

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.     

Agonist-induced subsensitivity of adenylate cyclase coupled with a dopamine receptor in slices from rat corpus striatum.

Incubation, for 30 min, of striatal slices with 10 microM dopamine, 10 microM apomorphine, or 10 microM SKF 38393 decreased dopamine-stimulated adenylate cyclase activity by 50-60%. This loss in dopamine-stimulated enzyme activity appears to be mediated by a persistent occupancy of recognition sites of the D-1 receptor because: (i) at 10 microM, SKF 38393, a selective D-1 receptor agonist, facilitates desensitization and Ly 141865, a selective D-2 receptor agonist, fails to elicit desensitization of dopamine-dependent adenylate cyclase; and (ii) preincubation with dopamine in the presence of 1 microM haloperidol but not 1 microM sulpiride curtails the desensitization of dopamine-dependent adenylate cyclase. In dopamine-desensitized striatal slices of the Kd for N-propylnorapomorphine binding is increased but the content of membrane-bound calmodulin and the activation of adenylate cyclase by NaF and cholera toxin are decreased significantly. In striatal slices incubated with dopamine for prolonged time periods the coupling of the GTP-binding protein with adenylate cyclase and dopamine recognition sites may be impaired and the content of membrane-bound calmodulin is decreased.     

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein.From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.     

Exploring the time dependence of serum clara cell protein as a biomarker of pulmonary injury in humans

We have previously demonstrated Clara cell protein (CC16) [secretoglobin 1A1] in serum to be a highly sensitive biomarker of altered lung epithelial permeability after ozone challenge. As a previous experimental study has indicated a diurnal variation in serum CC16 in humans, the aims of the present investigation were to confirm this observation and to attempt to model the diurnal variation in CC16 concentrations. In 18 healthy nonsmoking subjects, peripheral blood samples were drawn at six sampling points over a 15-h period and repeated twice within 3 to 4 weeks. A clear within-day variation was revealed in serum CC16 concentrations, falling significantly from baseline levels between the 11:30 am and 10:00 pm time points (p = 0.000). Furthermore, it was shown that this within-day variation was reproducible regardless of subject or day, enabling the diurnal variation in serum CC16 to be modeled and fitted a second-degree polynomial for the observed time span. In conclusion, the present data demonstrate a pronounced time-dependent diurnal variation in serum levels of CC16, which can be mathematically compensated for, when addressing the issue of an air pollution-induced effect on CC16 in field studies.