Biomedical subjects

P W Saphier

Publications and source records attributed to P W Saphier.

12 recordsLinked to original sources

PubMed2003

Identification of stimulatory and inhibitory inputs to the hypothalamic-pituitary-adrenal axis during hypoglycaemia or transport in ewes.

This study used the novel approach of statistical modelling to investigate the control of hypothalamic-pituitary-adrenal (HPA) axis and quantify temporal relationships between hormones. Two experimental paradigms were chosen, insulin-induced hypoglycaemia and 2 h transport, to assess differences in control between noncognitive and cognitive stimuli. Vasopressin and corticotropin-releasing hormone (CRH) were measured in hypophysial portal plasma, and adrenocorticotropin hormone (ACTH) and cortisol in jugular plasma of conscious sheep, and deconvolution analysis was used to calculate secretory rates, before modelling. During hypoglycaemia, the relationship between plasma glucose and vasopressin or CRH was best described by log10 transforming variables (i.e. a positive power-curve relationship). A negative-feedback relationship with log10 cortisol concentration 2 h previously was detected. Analysis of the "transport" stimulus suggested that the strength of the perceived stimulus decreased over time after accounting for cortisol facilitation and negative-feedback. The time course of vasopressin and CRH responses to each stimulus were different However, at the pituitary level, the data suggested that log10 ACTH secretion rate was related to log10 vasopressin and CRH concentrations with very similar regression coefficients and an identical ratio of actions (2.3 : 1) for both stimuli. Similar magnitude negative-feedback effects of log10 cortisol at -110 min (hypoglycaemia) or -40 min (transport) were detected, and both models contained a stimulatory relationship with cortisol at 0 min (facilitation). At adrenal gland level, cortisol secretory rates were related to simultaneously measured untransformed ACTH concentration but the regression coefficient for the hypoglycaemia model was 2.5-fold greater than for transport. No individual sustained maximum cortisol secretion for longer than 20 min during hypoglycaemia and 40 min during transport. These unique models demonstrate that corticosteroid negative-feedback is a significant control mechanism at both the pituitary and hypothalamus. The amplitude of HPA response may be related to stimulus intensity and corticosteroid negative-feedback, while duration depended on feedback alone.

Adrenal Cortex Hormones↗

PubMed1993

Purification and immunological characterization of pigeon serum butyrylcholinesterase. Implications on environmental monitoring and toxicological testing of birds.

Butyrylcholinesterase (EC 3.1.1.8) (BChE) was purified from pigeon serum to electrophoretic homogeneity by a four-step procedure involving blue sepharose CL-6B chromatography, ion exchange chromatography, procainamide affinity chromatography and gel filtration. An overall 2789-fold purification was achieved, with a final specific activity of 61.35 mumol/min/mg. The purified enzyme separated into two peaks when filtered through a column of Sephacryl S-300, a smaller peak containing the tetrameric form of BChE (C4) and a larger peak containing the monomeric form of BChE (C1). Native polyacrylamide gel electrophoresis (PAGE) of both peaks revealed single protein bands which coincided with esterase activity, with approximate M(r) values of 84,000 and 340,000, respectively. The C1 monomer represented 85-90% of the activity found in the pigeon serum. It is not clear whether this polymorphism of BChE in vertebrates contributes to the wider inter-individual variations observed in xenobiotics elimination kinetics and in the response to the pharmacological and toxic effects of pesticides. PAGE of the monomeric form of the enzyme in the presence of sodium dodecyl sulphate showed only one protein band with a M(r) of 84,000, while that of the tetrameric form revealed two bands, a major protein band (84,000) and a minor band (170,000), representing the monomer and the dimer of the dissociated tetrameric BChE enzyme under reducing conditions. Highly specific polyclonal antibodies were raised in rabbits against the purified enzyme. These antibodies cross-reacted with other avian BChEs, a criterion which make them useful for the immunopurification of other BChEs from different species as well as for biomonitoring and toxicological studies on the role of esterases as an indicator of avian exposure to organophosphorous pesticides.

Animals↗

PubMed1993

Thymus-pituitary interactions during ageing.

We had previously shown that homoeostatic thymus hormone (HTH), an H2A-H2B histone dimer, possesses an age-dependent adrenostimulatory activity in vivo. It was therefore of interest to investigate the adrenocorticotropin (ACTH)-releasing activity of histones and other related preparations on perifused pituitary cells from young (2-4 months) and mature (16-18 months) rats. Histones and protamines gave a positive interference with the immunoradiometric assays (IRMA) for ACTH and corticotropin releasing hormone (CRH) whereas the histone fragment MB35, as well as nucleohistone and nucleoprotamine complexes did not. The latter three substances induced a significant ACTH response in pituitary cells from young and, to a lesser extent, mature rats. Our results suggest that nucleoprotein complexes released into the extracellular milieu may act as hypophysiotropic signals. On the other hand, the responsiveness of pituitary cells to these signals seems to decrease with the age of the donor.

Adrenocorticotropic Hormone↗

PubMed1993

Elevated levels of N-terminal pro-opiomelanocortin peptides in fetal sheep plasma may contribute to fetal adrenal gland development and the pre-parturient cortisol surge.

Parturition in sheep is initiated by a rapid rise in fetal plasma cortisol. There is some controversy as to the exact nature of the drive for this pre-partum cortisol surge and it is thought that factors other than ACTH may act in concert to stimulate the development of the fetal adrenal gland. We have investigated the concentrations of ACTH and other peptides derived from pro-opiomelanocortin (POMC) in the circulation of fetal sheep during the final part of gestation, using specific 2-site immunoradiometric assays. The expected rise in fetal cortisol was seen with an 880% (p < 0.01) increase in concentration of this hormone between the initial measurement period (110-119 days gestation) and the final period (139-147 days). Fetal plasma ACTH increased less dramatically (137%; p < 0.03) during this time. The most surprising finding was the presence of very high relative concentrations of the N-terminal POMC peptide N-POMC(1-77) in the fetal circulation. Initially the concentration was 289 +/- 66 pmol/l compared to ACTH concentrations of 6.4 +/- 0.8 pmol/l. In the final week of gestation N-POMC(1-77) levels, although still high, had declined to 188 +/- 35 pmol/l (ACTH having increased to 13.7 +/- 2.2 pmol/l). Fetal plasma 3 yen-MSH was found to increase towards the end of gestation when the concentration of N-POMC(1-77) was declining, suggesting some cleavage of the latter. We postulate that the N-POMC(1-77) and its fragments, acting in concert with ACTH, play a vital role in stimulating the development of the fetal adrenal cortex and provide the additional drive to the adrenal gland required to stimulate parturition.

Adrenal Glands↗

PubMed1992

A comparison of the clearance of ovine and human corticotrophin-releasing hormone (CRH) in man and sheep: a possible role for CRH-binding protein.

A specific binding protein for human corticotrophin-releasing hormone (hCRH), which does not bind to the ovine hormone (oCRH), has recently been demonstrated in human plasma. No such binding protein has been found in sheep plasma. We have investigated the half-life of human and ovine CRH in man and in sheep. Peptides were measured directly in plasma with two-site immunoradiometric assays, as these assays are unaffected by the presence of inactivated peptide fragments. In man, the half-life of hCRH (30.5 +/- 3.3 min; mean +/- S.E.M.) was significantly (P less than 0.001) less than that of oCRH (42.8 +/- 6.4 min). In sheep, there was no significant difference between the half-life of hCRH (46.5 +/- 7.2 min) and that of oCRH (39.8 +/- 10.1 min); these half-lives were also significantly (P less than 0.001) longer than that of hCRH in man. One possible explanation for the shorter half-life of hCRH in man is that the clearance of hCRH is enhanced by CRH-binding protein, although other binding proteins often have the opposite effect. Peak ACTH and cortisol responses occurred earlier in sheep than in man, although no differences were found in the response times to oCRH or hCRH within either species. The responses were more sustained in sheep than in man, and the previously reported biphasic response was only seen in some of the sheep and not in man. Absolute responses to either peptide were greater in sheep than in man; however, in man an 8.1-fold rise in ACTH was measured in response to oCRH, while hCRH gave a significantly (P = 0.043) smaller 4.4-fold response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1991

Biological properties of synthetic human parathyroid hormone: effect of deamidation at position 76 on agonist and antagonist activity.

Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.

Adenylyl Cyclases↗

PubMed1990

Fluoride therapy and parathyroid hormone activity in osteoporosis.

1. To determine the relationships between parathyroid hormone activity and long-term sodium fluoride therapy in osteoporosis, cytochemical bioassays (for biologically active parathyroid hormone) were performed in 22 osteoporotic control patients and in 18 patients after 15 +/- 10 months of treatment (60 mg of sodium fluoride daily). Ten patients were studied longitudinally by repeated metabolic balances and were therefore common to both groups. All patients were receiving mineral supplements. 2. Cross-sectional data showed a fourfold mean increase in biologically active parathyroid hormone on fluoride treatment (P less than 0.005) together with a 51% increase in serum alkaline phosphatase (P less than 0.005). Longitudinal data showed, in addition, a significant increase in the calcium balance of 2.4 +/- 1.2 (SEM) mmol daily (P less than 0.05) and the development of a positive phosphorus balance (P less than 0.02). 3. Fluoride-treated patients were then analysed in two groups according to the level of biologically active parathyroid hormone. Thirty-two per cent of values were above the upper limit of normal (18 pg/ml). The mean serum alkaline phosphatase level in this group showed no elevation above that of the control patients, the overall rise being accounted for entirely by patients with normal levels of biologically active parathyroid hormone. High levels of biologically active parathyroid hormone were also associated with relative hypophosphataemia (P less than 0.01), relative hypercalciuria (P less than 0.05) and an increased urine/faecal calcium ratio (P less than 0.025). 4. Results show that long-term fluoride and calcium therapy increase biologically active parathyroid hormone in osteoporosis and that excessive parathyroid hormone activity may account for certain features of the refractory state.

Adult↗

PubMed1990

Corticotropin-releasing hormone (CRH)-binding protein: reduction in the adrenocorticotropin-releasing activity of placental but not hypothalamic CRH.

This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.

Adrenocorticotropic Hormone↗

PubMed1990

The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety.

We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.

Animals↗

PubMed1988

Fluoride therapy in osteoporosis: acute effects on parathyroid and mineral homoeostasis.

1. Acute metabolic effects of sodium fluoride therapy were analysed among 41 osteoporotic patients already receiving large calcium supplements, 33 of whom underwent simultaneous metabolic balance studies. 2. Mean serum calcium fell transiently within 24-48 h by 0.03 +/- 0.07 (SD) mmol/l (P less than 0.01) and phosphorus by 0.06 +/- 0.08 (SD) mmol/l (P less than 0.001). In a subgroup, ionized calcium fell and biologically active parathyroid hormone (bio-PTH) rose more than fivefold (P less than 0.01). Urine calcium rose after an insignificant fall. 3. Pretreatment calcium and phosphorus balances were significantly positive and did not change overall during the first 8 days of treatment. However, on analysing balances in two groups relative to serum changes, in patients whose serum levels changed least sodium fluoride increased faecal calcium (P less than 0.025) and phosphorus (P less than 0.01) and reduced calcium balance (P less than 0.01), giving a mean balance difference between the two groups of 2.1 mmol daily (P less than 0.001). 4. Very small changes in serum levels therefore indicate well-marked metabolic responses: sodium fluoride acutely stimulates bio-PTH activity and must also enhance mineral uptake from circulation into tissue(s). By separate and opposing action(s) it inhibits intestinal calcium and phosphorus absorption, predominantly in those whose serum levels remain stable. All these effects may be relevant to long-term therapeutic results.

Aged↗

PubMed1987

Studies on the measurement of 25-hydroxy vitamin D in human saliva.

A competitive protein-binding assay for 25-hydroxy vitamin D (25-OHD) in saliva has been established by adaptation of that previously described for 25-OHD in serum (Fairney et al. 1979). Random values of salivary 25-OHD in patients attending hospital for venesection showed a wide range of results (105-1000 pg/ml, n 55). These values corresponded to 1.2% of the total serum values with which they showed a significant relation (r 0.45, P less than 0.001). There was no relation between salivary 25-OHD and measured serum free 25-OHD in eighteen pairs of saliva and serum studied. Studies in two individuals showed that salivary 25-OHD values varied throughout the day and that a vitamin D load (19.5 micrograms), given as pickled herrings at lunch, produced a marked rise in 25-OHD values 5-8 h later. Diurnal profile studies of salivary 25-OHD in Caucasian and Asian 11-year-old male schoolchildren showed lower values in Asian children eating a vegetarian diet, and a significant variation with time and ethnic group (P less than 0.001). It is concluded that 25-OHD is present in saliva and that the values vary throughout the day. The values obtained may relate to dietary intake of vitamin D and the subject's ethnic origin.

Calcifediol↗

PubMed1987

PTH bioactivity in osteoporosis.

The cytochemical bioassay for parathyroid hormone was used to measure the levels of PTH bioactivity in normal elderly subjects and patients suffering from idiopathic or postmenopausal osteoporosis. In normal subjects, PTH bioactivity did not increase with age. In the osteoporotic patients levels of bioPTH discriminated between two populations, one with normal levels and the other with elevated levels. In both these groups the PTH bioactivity was neutralised by the addition of a PTH antiserum. The recovery of the bioactivity of PTH added to the plasma samples was significantly higher in the plasmas from patients with elevated levels of bioPTH. Levels of immunoreactive PTH also discriminated between the two groups of osteoporotic patient although no serum parameters indicative of increased PTH were detectable. However, patients with the high levels of bio- and immunoPTH had a lower incidence of fracture within the previous 12 months.

Adult↗