Cellular actions of inositol phosphates and other natural calcium and magnesium chelators.

Naturally occurring chelators of Ca2+ and Mg2+ have largely been unrecognized due to their low binding affinities. They include carbohydrate and cyclitol phosphates, nucleotides and nucleic acids. The calciotrophic inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 form chelates within the range of Ca2+ concentrations found in biological systems. As well as being a likely source of experimental artifact where these compounds have been investigated at unphysiological cation concentrations, chelation may have important physiological roles. The autoregulation of Ca2+ entry into the cell cytosol is one, whereas the coupling of chelation with enzyme or receptor interactions offers a general mechanism for divalent cation control of diverse biological processes. Inositol monophosphate 1-phosphatase and inositol polyphosphate 1-phosphatase are two related enzymes which may conform to this mechanism. If so, it would provide a possible explanation for their sensitivity to divalent cations and for their non-competitive inhibition by lithium ion.

The urinary excretion of glucosiduronates of cortisol and cortisone.

Cortisol and cortisone glucosiduronic acids were synthesised in a 14C-labelled from and utilised in a double-isotope derivative procedure for the analysis of cortisol glucosiduronate (FG) and cortisone glucosiduronate (EG) in human urine. Normal adults were found to excrete between 16 and 100 mug/24 h of FG (n = 14) and between 55 and 120 mug/24 h of EG (n = 15). Elevated values were observed in subjects with Cushing's syndrome and following ACTH stimulation. Abnormal excretion was noted in one patient with hepatic cirrhosis and in one case of cholestatic jaundice. The ratio FG/EG was markedly increased after ACTH stimulation and, in the normal group, was positively correlated to a highly significant degree (P less than 0.001) with FG excretion. These two observations suggest that EG excretion is less sensitive than FG excretion to variations in cortisol production.

Metabolism of cortisol-21 glucosiduronate in man.

The metabolism of cortisol-21-glucosiduronate has been studied in two subjects. Urinary excretion was about 65% in both subjects, with cortisol glucosiduronate as the principal metabolite accompanied by small amounts of 11 beta, 17,21-trihydroxypregnane-3,20-dione (THF) glucosiduronate. The absence of the other normal metabolites of cortisol [17,21-dihydroxypregnane-3,11-20-trione (THE), 3 alpha, 11 beta, 17, 20xi, 21-pentahydroxypregnane, and 3 alpha, 17, 20xi, 21-tetrahydroxypregnan-11-one] indicates that negligible hydrolysis at C-21 occurred and that an unoccupied C21 position is required for oxidation at C-11 and reduction at C-20. The limited conversion to THF suggests that a pathway through cortisol-21-glucosiduronate does not contribute significantly to the differences in specific activity of urinary THF and THE observed after administration of labeled cortisol.

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells.

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.

Interaction of the mouse thyrotrophin receptor with thyrotrophin binding inhibitor immunoglobulins.

The species-specificity of thyrotrophin binding inhibitor immunoglobulin (TBII) for the thyroid TSH receptor was investigated using a preparation of thyroid plasma membranes (TPM) from propylthiouracil-treated mice, as well as from human glands. The interest in the mouse arose from its use as the bioassay animal for the long-acting thyroid stimulator (LATS). A comparison was made of the response in the two radioreceptor assays of serum immunoglobulins from ten normal subjects and twenty patients with Graves's disease, who had also been selected to have positive TBII activity in the assay based on human TPM. All the specimens from the patients with Graves's disease had detectable TBII activity in the mouse radioreceptor assay, inhibiting the binding of 125I-labelled TSH to a greater extent than did any of the specimens from normal subjects. There was evidence for a minor degree of species-specificity, since at least one of the specimens from the Graves' disease group had unexpectedly high activity in the assay based on mouse TPM and another had unexpectedly weak activity in that assay. However, this specificity appeared to be unrelated to the presence or absence of LATS. The effect of LATS on the response of serum immunoglobulins in the mouse radioreceptor assay was tested using nine patients with Graves's disease who had undetectable serum LATS and another eight patients with Graves's disease whose serum gave a positive LATS response. These patients had also all been selected to have positive TBII activity in their serum, as determined with human TPM. All samples from each of the LATS-positive and LATS-negative subgroups gave a positive TBII response in the radioreceptor assay based on mouse TPM, and there was extensive overlap between the individual values for the two subgroups. It is concluded that the failure of some TBII-positive serum immunoglobulins to stimulate the mouse thyroid gland and produce a positive LATS response is not due to species-specificity at the level of receptor binding.

The free androgen index is not valid for adult males.

The Free Androgen Index (FAI) was initially proposed as a measure for assessing the circulating testosterone availability in female hirsutism. The extension of its use, by a number of investigators, to males has not been formally justified. An analysis of its derivation from the Law of Mass Action reveals an implied assumption that the binding capacity of sex hormone binding globulin should greatly exceed the concentration of its ligand testosterone. This does not hold in adult males for whom the use of FAI is therefore inappropriate. A comparison of FAI and free-testosterone (determined by centrifugal ultrafiltration) yielded a correlation coefficient (r) of 0.858 for 20 adult females but only 0.435 for 19 adult males.

Use of the Becton-Dickinson urine culture tube with the Abbott MS-2 urine screening system.

Urine specimens were obtained from 312 obstetric outpatients by sterile midstream technique and aliquots placed in both Becton-Dickinson urine culture tubes and sterile conventional tubes. Quantitative cultures were made from each tube, and each tube was screened for bacteria with the Abbott MS-2 urine screening system. The time required to detect bacteriuria was recorded for both specimens. Isolates from specimens containing greater than or equal to 50,000/ml gram-positive cocci or greater than or equal to 100,000/ml gram-negative bacilli were identified and antimicrobial susceptibility tests performed. Delayed (24 hr) quantitative cultures were done from Becton-Dickinson tubes. By these criteria, 124 urine specimens were positive in both conventional and Becton-Dickinson tubes. Escherichia coli (n = 72), Klebsiella pneumoniae (n = 20), Enterobacter cloacae (n = 8), Proteus mirabilis (n = 4), group B streptococcus (n = 12), and enterococcus (n = 8) were isolated. Time for detection of positive urine samples was similar in both types of tubes. Delayed cultures had significant numbers of false-positive results. Antimicrobial susceptibility results did not appear to be influenced greatly by Becton-Dickinson tube transport. The MS-2 cannot adequately discriminate cultures containing less than 50,000 colony-forming units/ml of urine. The Becton-Dickinson tube appears to be compatible for use with the MS-2 for purposes of screening for bacteriuria.

Susceptibility in vitro of Nocardia species to antimicrobial agents.

Fifty-four clinical isolates of Nocardia spp. were tested in vitro for susceptibility to several antimicrobial agents. Of these, 89% were susceptible to ciprofloxacin, 86% to imipenem, 85% to fusidic acid, and 71% to cefotaxime. Some of the agents may be suitable alternative or adjunctive drugs to sulfonamides and aminoglycosides for chemotherapy of Nocardial infections.

P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors.

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.

Mitogenic effect of lithium in FRTL-5 cells can be reversed by blocking de novo cholesterol synthesis and subsequent signal transduction.

Lithium therapy is the therapeutic mainstay for bipolar disorder and has been associated in the thyroid with euthymic goiter, hyper and hypothyroidism as well as thyroid autoimmune disease. The FRTL-5 cell line is a well known model of thyroid cell physiology, where lithium has been shown to increase 3H-thymidine uptake at concentrations of 2 mM. This mitogenic effect was not associated with adenylate cyclase as measured by cyclic adenosine monophosphate (cAMP) production. The de novo synthesis of cholesterol is an important signal transduction pathway in FRTL-5 cells, where newly synthesized Rho GTPase is geranylgeranylated, enabling membrane localization of the G-protein and subsequent G1 to S-phase transition, resulting from extracellular stimulation. Here we confirm lithium mitogenicity at therapeutically relevant concentrations (1 mM) and demonstrate a lithium-associated accumulation of FRTL-5 cells in S-phase of the cell cycle. These effects could be abolished by Pravastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the rate-limiting enzyme in the formation of intermediates (de novo cholesterol synthesis) required for G-protein prenylation. Pravastatin, similar to lithium, showed no effect on cAMP production either under basal or thyroid stimulating hormone (TSH)-stimulated conditions indicating that de novo cholesterol synthesis is not involved with adenylate cyclase. The inhibitory effect of pravastatin could be overcome by reinitiating de novo cholesterol synthesis. This was achieved by the addition of the cell permeable, first metabolite (mevalonate) after HMG-CoA, which allowed the cycle to continue, leading eventually to protein prenylation, despite the presence of Pravastatin. These novel findings demonstrate lithium involvement in de novo cholesterol synthesis and G-protein prenylation, an important signal transduction pathway in FRTL-5 cells.

The Fischer rat thyroid cell line FRTL-5 exhibits a nondiploid karyotype.

The FRTL-5 cell line is a stable thyroid cell line derived from the thyroid gland of the Fischer rat under defined culture conditions, which has been widely adopted as a model system for the study of thyroid cell function and for bioassay. While characterizing by flow cytometry FRTL-5 cells that were supplied to this laboratory by ATCC (American Type Cell Collection), we discovered that the cells (ATCC CRL8305) were not diploid, having approximately twofold the DNA content relative to a diploid control. The increase in DNA content also applied to cells originally supplied by the ATCC (described as passage 14) that when counted in metaphase had a modal chromosomal count of 84, indicating tetraploid status, double the expected 42 of a diploid rat cell. In a private communication, the ATCC confirmed these findings which nevertheless are contrary to previous literature reports where they were reported to be diploid. Tetraploid cells are less sensitive to thyrotropin (TSH) as measured by cyclic adenosine monophosphate (cAMP) production, compared with diploid cells (p = < 0.001). Despite similar 3H-thymidine uptake in 0.2% fetal calf serum, tetraploid cells show increased 3H-thymidine uptake in 5% fetal calf serum in the absence of TSH (p = 0.001). The origin of these chromosomal changes is unclear, but these findings must raise doubts regarding the suitability of the tetraploid FRTL-5 cell line as a model for studies of human or animal thyroid physiology.

Pregnanetriolone, a normal steroid metabolite: its excretion by normal, Cushing's syndrome and congenital adrenal hyperplasia subjects.

Synthesis of 3H-pregnanetriolone permitted the estimation of pregnanetriolone in urine with a sensitivity in excess of most previous claims. A good correlation (r = +0.97) was obtained between the values from gas liquid chromatography and those of a double isotope derivative method. In contrast to previous reports, these methods indicated that pregnanetriolone is excreted by normal adults. Urinary pregnanetriolone levels were 18-59 mug/24hr for normal subjects, 35-290mug/24hr in Cushing's syndrome and 250-7000 mug/24hr with congenital adrenal hyperplasia. It is concluded that pregnanetriolone is a normal steroid metabolite and its occurrence in Cushing's syndrome does not necessary indicate an abnormal steroid biosynthetic pathway.

Longitudinal study of plasma ACTH and cortisol in very low birth weight infants in the first 8 weeks of life.

There are few published data on plasma ACTH and cortisol in very low birth weight (VLBW) infants beyond the first week of life. We therefore measured plasma ACTH and cortisol longitudinally in 25 infants (mean birth weight 1025 g, mean gestational age 28 weeks) at 1, 2, 4 and 8 postnatal weeks to document normative values for infants not receiving dexamethasone. We also examined the influence of clinical state and dexamethasone treatment on plasma ACTH and cortisol levels. Median plasma ACTH increased significantly with advancing postnatal age from 1 week to 8 weeks (21.0 vs. 40.0 ng/l; P = 0.01) but did not correlate with postconceptional age. Median plasma cortisol decreased significantly with advancing postnatal age from 1 week to 8 weeks (216 vs. 50 nmol/l; P = 0.001) and correlated inversely with postconceptional age (P = 0.004). At 8 weeks infants who were clinically well (n = 6) had lower plasma ACTH values compared with sick (n = 6) infants (median: 37.0 vs. 63.5 ng/l; P = 0.033). Plasma ACTH did not correlate with clinical state at 1, 2 and 4 weeks. At none of the postnatal ages studied was plasma cortisol influenced by the degree of sickness. Five infants received dexamethasone to assist weaning from mechanical ventilation. Their median plasma ACTH level, at 8 weeks, was significantly lower than that of the 12 infants who did not receive dexamethasone (11.0 vs. 40.0 ng/l; P = 0.0006). Plasma cortisol was not significantly influenced by dexamethasone treatment (P = 0.27). These data provide further information on the evolution of adrenocortical function in VLBW infants in the first months of life.

The prenatal determination of fetal sex: amniotic fluid testosterone as a preliminary screening test.

Amniotic fluid testosterone levels were measured by radioimmunoassay without chromatography on 101 specimens obtained at amniocenteses between 15 and 19 wk gestation. For the male fetus, the amniotic fluid testosterone level of 553 +/- 23 pmol/l (mean +/- SE) was significantly higher (P less than 0.0005) than the concentration found for the female fetus (206 +/- 9 pmol/l). There was an overlap of the ranges 74-1120 pmol/l for the male and 122-399 pmol/l for the female fetuses. Amniotic fluid testosterone levels above 400 pmol/l were observed in 84% of the male and in none of the female fetuses. The method allowed determination of testosterone levels within 8 h. It is concluded that amniotic fluid testosterone measured by radioimmunoassay without chromatography is a rapid and effective preliminary screening test for the prenatal diagnosis of fetal sex.

The biological relevance of the binding of calcium ions by inositol phosphates.

The binding of Ca2+ (chelation) by myo-inositol polyphosphates at pH 7.0 was studied using a Ca(2+)-sensitive electrode. Glucose 6-phosphate (used as a model for a monophosphate) bound Ca2+ with an affinity of 152 +/- 31 liters/mol and a molar ratio of 0.94 +/- 0.02. Inositol 3,4-bisphosphate, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol hexakisphosphate showed affinities of 9.0 +/- 2.1 x 10(3), 6.3 +/- 1.5 x 10(3), 6.2 x 10(4), and 1.92 +/- 0.47 x 10(5) liters/mol, respectively, and molar ratios of 0.92 +/- 0.49, 0.95 +/- 0.10, 0.75, and 2.5 +/- 0.5. In general, the affinity increased with the number of phosphate substituents on the inositol ring, although the stereochemistry is also expected to be important. This suggests that for the physiologically relevant inositol phosphates (tris-, tetrakis-, pentakis-, and hexakis-) half-maximal Ca2+ binding will occur in the Ca2+ concentration range of approximately 5 x 10(-6) to 2 x 10(-4) M. This range lies between the basal intracellular and the fee extracellular Ca2+ levels (10(-7) and 10(-3) M), respectively, and may therefore be of physiological importance. Chelation provides a possible simple explanation for the inhibition by Ca2+ of inositol 1,4,5-trisphosphate binding to its receptor in rat cerebellum and other tissues. It may also have a role in limiting inositol phosphate-mediated increases in intracellular Ca2+.

Computerized method for validating laboratory reference ranges for triiodothyronine and thyroxin immunoassays.

We used a computer-based method to help validate the reference ranges of assays for triiodothyronine (T3) and thyroxin (T4). A retrospective search of a database of laboratory results for the previous six months identified all patients with apparent euthyroid status, as defined by methods independent of the immunoassay under review. A computer-generated reference group (CGR Group) of 2001 records had a gaussian distribution of T4 values and a reference range (mean +/- 2 SD) of 56-161 nmol/L, compared with the supplier's suggested range for euthyroid subjects (58-148 nmol/L) and an in-house range of 60-144 nmol/L for a group of 97 normal subjects. A similar CGR Group of 1902 records gave a reference range for T3 of 0.7-2.1 nmol/L (manufacturer's range 0.8-2.8; normal subjects 0.8-2.2). An attempt to devise a reference range for thyrotropin failed when we found that its concentration in the population of patients with normal values for thyroid hormones was distributed differently from that in the normal population. The method is intended to be used in addition to conventionally derived ranges based on results for healthy subjects. It allows the laboratory to conveniently verify the reference ranges for T3 and T4 assays at regular intervals by using very large samples with appropriate age, sex, and weight distribution, drawn from the population of patients' samples submitted for analysis.

Reaction coupling of chelation and antigen binding in the calcium ion-dependent antibody binding of cyclic AMP.

Polyclonal antibodies, raised against cyclic AMP (cAMP) by the immunization of animals with a 2'-O-succinyl cAMP/bovine albumin conjugate, have been reported to be dependent upon the presence of calcium ion (Ca2+) for antigen binding. They also exhibit a major "bridge" effect whereby 2'-O-succinyl and 2'-O-acetyl derivatives are bound more avidly than the parent nucleotide. Since cAMP and these derivatives bind Ca2+ very weakly, they do not present substantially in the chelated form over the range of Ca2+ concentrations used. Thus direct antigen modification is excluded as an explanation for the observed ion dependence of the reaction. Instead, we propose a mechanism based on reaction coupling. The actual antigens are the Ca2+ chelates of these nucleotides, whose formation in the absence of antibody is rapid but not favored (as indicated by their weak association constants). When antibody is added, the chelates act as transient intermediates whose concentration remains low but which is replenished as they are consumed by antibody. The coupled reaction is driven by the antibody-antigen step which occurs more slowly but with a substantial gain in free energy. The reaction is limited by the availability of Ca2+. It also appears that the rabbit antibody-forming cell responds preferentially to the Ca(2+)-bound form of the 2'-O-succinyl cAMP/bovine albumin conjugate which may appear to be more "foreign" than the unbound form of the hapten containing the ubiquitous nucleotide cAMP.

The urinary excretion of cortisol and cortisone glucosiduronates in pregnancy.

The urinary excretions of cortisol and cortisone glucosiduronates were measured at monthly intervals in three normal pregnancies by a double isotope dilution derivative method. The excretions were mostly within the normal range, but some were above the normal range suggesting that these excretions may increase in some pregnancies.

Thyroid stimulating immunoglobulin in Graves' disease.

There is excellent presumptive evidence that an IgG plays an aetiological role in the development of hyperthyroidism in Graves' disease but available methods for detecting thyroid stimulating immunoglobulins (TSI) are still far from satisfactory. They show considerable variation in specificity, sensitivity and precision, and comparison of the experimental data, obtained with these various methods, is difficult. A need exists for an in vitro TSI assay which is based upon the propensity of IgG molecules to stimulate the thyroid gland. Because a complex chain of biochemical events is involved, including binding to IgG to the cell membrane receptor, release of cyclic AMP, organification of iodide, hydrolysis of iodoproteins and secretion of thyroid hormones, it is not yet clear which step should be monitored to obtain the best index of thyroid stimulating activity. Although TSI and related assays appear to be of limited value in the primary diagnosis of Graves' disease, they offer some assistance to the clinician in the following situations: 1. Prediction of relapse in Graves' disease patients who have been rendered euthyroid with antithyroid drugs. 2. Identification of patients with ophthalmic Graves' disease. 3. Prediction of neonatal hyperthyroidism in thyrotoxic pregnancies.