Joyce B. Baumann

Nerve growth factor induces plasma extravasation in rat skin

The intradermal application of both mouse and bovine nerve growth factor induced dose-dependently increased vascular permeability as indicated by Evans blue extravasation. Plasma extravasation by nerve growth factor was markedly reduced by capsaicin pretreatment or a combination of H1- and H2-histamine antagonists. The finding that biologically inactive nerve growth factor failed to elicit protein leakage indicates that plasma extravasation by nerve growth factor is a characteristic of the biologically active molecule.

Effect of melanin concentrating hormone on pigment and adrenal cells in vitro

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.7 and with propargylglycine by a factor of about 7. Linear, Cys5,14-Acm-protected MCH was a full agonist of MCH but with a 345-fold lower potency. Iodinated MCH showed similar, low activity. In tetrapods, salmonid MCH and its analogs displayed only marginal pigment dispersion at concentrations greater than 10(-5) M. Alkali-treatment of MCH increased the pigment-dispersing potency by a factor of about 30 whereas the activity for pigment aggregation in the grass carp was destroyed. At high concentrations (10(-6), 10(-5) M) MCH also stimulated tyrosinase activity in B-16 mouse melanoma cells but did not modify the effects of alpha-MSH in this system. By contrast, when tested on rat adrenal glomerulosa cells, salmonid MCH had no effect alone but at a concentration of greater than 10(-10) M it slightly reduced corticosterone production by an alpha-MSH concentration of 10(-7) M. Aldosterone production was not affected and MCH did not influence the response to ACTH.

Growth hormone in urine: development of an ultrasensitive assay applicable to plasma and urine

A radiometric assay for human growth hormone (HGH) was developed based on a polyclonal goat anti-HGH antiserum covalently coupled to nonsedimenting polyacrylamide particles. HGH can be specifically immunoextracted from sample volumes of up to 10 ml. Subsequently, bound HGH is identified and quantitatively measured by a 125I-labelled monoclonal anti-HGH antibody. The assay is insensitive to plasma proteins from 10 to greater than 90%, to changing NaCl and urea molarities and to pH ranges from 6 to 8. The sensitivity in the second incubation is 2 pg/tube, corresponding to a maximum sensitivity of 300 fg/ml of a sample volume of 10 ml (urine) or of 40 pg/ml, if a volume of 50 microliter (plasma) is assayed. In healthy children, a mean HGH excretion of 6.5 ng/24 h was found with a large interindividual range from undetectable to 37.4 ng. An important intraindividual night-to-night variation of HGH excretion was found in several subsequent first morning void samples in healthy children. The mean excretion in 13 HGH-deficient children was 0.9 ng/24 h off therapy and increased to a mean of 6.9 ng/24 h on therapy. In acromegalic patients, the excreted HGH amounted to 73-208 ng/24 h. Preliminary results suggest that the ultrasensitive assay applied to plasma and urine could be a considerable improvement of diagnosis and follow-up of disorders of HGH secretion.

Quantitative analysis of ACTH-immunoreactive cells in the anterior pituitary of young spontaneously hypertensive and normotensive rats

SummaryACTH-immunoreactive cells in the anterior pituitary of 4-week-old spontaneously hypertensive rats (SHR) were studied with immunocytochemical and morphometric techniques. The results were compared with data from age-matched normotensive Wistar-Kyoto rats (WKY). No significant differences were found in volume density and average size of ACTH-immunoreactive cells between these two strains. However, SHR showed a significantly larger anterior lobe (2 P < 0.01) than WKY, indicating that the total number of ACTH-immunoreactive cells in the anterior pituitary is greater in SHR than in WKY. These data are in agreement with radioimmunological determinations showing a significantly elevated content (2 P < 0.01) but only a moderately higher concentration (0.05 < 2 P < 0.10) of ACTH in the anterior pituitary of SHR as compared to WKY. The present results suggest an enhanced availability of ACTH in the anterior pituitary of 4-week-old SHR, a fact which could explain the markedly enhanced stress-induced release of ACTH previously found in these animals. This study further supports the hypothesis that, among other factors, an instability of the hypothalamo-pituitary-adrenal axis may contribute to the development of genetically programmed hypertension.

Human growth hormone in urine: development of an ultrasensitive radiometric assay

An immunoradiometric assay for human growth hormone (HGH) has been developed which has a detection limit of 1ng/l and can measure HGH in unextracted urine from normal children and adults. The assay is based on a two-step procedure, using a solid-phase goat-anti-HGH immunosorbent for immunoextraction and [125I]-labeled monoclonal HGH-antibody for detection and quantification. The assay is not affected by urea, NaCl or changes of pH from 5–8. The mean urine HGH concentration in normal children is 6.78±7.6 (SD) pg/ml, in patients with HGH-deficiency 1.3±0.9 pg/ml which increases to 11.7±13.4 pg/ml on the day of growth hormone injection.

Long-Term Effects of Betamethasone on Blood Pressure and Hypothalamo-Pituitary-Adrenocortical Function in Spontaneously Hypertensive and Normotensive Rats

An enhanced hypothalamo-pituitary-adrenocortical (HPA) activity has been described during onset of elevated blood pressure in spontaneously hypertensive rats (SHR). An instability of the HPA axis could thus contribute to the development of hypertension in these animals. Glucocorticoid effects on blood pressure and HPA function were studied therefore in SHR and normotensive Wistar-Kyoto (WKY) and Wistar rats. Beginning at 4 weeks of age, the rats were treated with 0.1 and 0.5 microgram betamethasone per milliliter drinking water for 7 weeks. SHR and WKY responded with a significant elevation in average blood pressure. In SHR, mean blood pressure rose from 181.4 +/- 3.9 (mean +/- SEM) to 203.1 +/- 2.8 mm Hg in response to the lower dose of betamethasone and to 209.2 +/- 4.0 mm Hg in response to 0.5 microgram betamethasone per milliliter drinking water. In WKY, blood pressure increased from 134.4 +/- 3.3 to 148.2 +/- 3.0 and 157.9 +/- 4.5 mm Hg in response to the lower and higher dose of betamethasone, respectively. No significant effect was seen in Wistar rats, where the mean blood pressure values changed insignificantly from 133.8 +/- 2.1 to 136.3 +/- 3.2 and 135.6 +/- 2.4 mm Hg. Stress-induced secretion of corticosterone was significantly suppressed in a dose-dependent manner in all three strains. Stress-induced secretion of adrenocorticotropin was markedly reduced by 0.5 microgram betamethasone per milliliter in SHR and by both doses in WKY. No significant effect, however, was seen in Wistar rats. A predisposition to the hypertensiogenic actions of glucocorticoids was found therefore in SHR and WKY, but not in Wistar rats.

MSH Receptors and the Response of Human A375 Melanoma Cells to Interleukin-1β

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) has been shown to be an inhibitory factor in many immunologic and inflammatory processes involving the cytokine interleukin-1 (IL-1). As the mechanism of the interaction between IL-1 and alpha-MSH at the receptor level is unknown, we have studied the role of MC1 melanocortin receptors in two variants of the human melanoma cell line A375 differing in their sensitivity to the cytostatic effects of IL-1 beta. Both IL-1 sensitive (A375r-) and resistant cells (A375r+) carry specific high affinity receptors for IL-1, albeit their concentration is 10-fold higher in A375r+ cells. In A375r- cells, MC1 receptors are absent or below the level for reliable detection in the binding assay. Conversion of A375r- to A375r+ cells by prolonged culture in medium not depleted of endotoxin led to the appearance of MC1 receptors (KD 0.4 +/- 0.123 nmol/l; 608 +/- 134 receptors/cell). Stable transfection of A375r- cells with the human MC1 receptor did not, however, render them resistant to the cytostatic effect of IL-1 beta on concomitant treatment with alpha-MSH or result in the production of IL-6 on treatment with IL-1 beta. Therefore, the presence of MC1 receptors on the surface of A375 cells or their binding to alpha-MSH does not seem to be a factor in cytokine resistance or IL-6 secretion. No interaction between IL-1 beta and alpha-MSH could be demonstrated at the cellular level in this melanoma cell line.

Inhibition of the ACTH Adrenal Response to Stress by Treatment with Hydrocortisone, Prednisolone and Dexamethasone in the Rat

16- and 4-week-old intact and adrenalectomized rats have been treated with different doses of the three glucocorticoids hydrocortisone, prednisolone and dexamethasone by gavage. The delayed feedback effect on plasma ACTH and corticosterone response to an ether stress have been assessed. Almost complete suppression of corticosterone response 20 min after an ether stress and an ACTH suppression to 20% of control values 5 min after an ether stress were observed with 25 micrograms of dexamethasone, 10 mg of prednisolone and 20 mg of hydrocortisone. Although the percent inhibition of corticosterone and ACTH response to stress was comparable, a striking dissociation of the ACTH and corticosterone release was observed in terms of absolute concentrations. A mean ACTH concentration of 462 ng/l after 25 micrograms of dexamethasone was measured together with a barely measurable corticosterone concentration of 3 micrograms%. Similarly, after 10 mg of prednisolone, the mean ACTH concentration was 404 ng/l, whilst the mean corticosterone concentration was 3 micrograms%. This dissociation demonstrates that the corticosterone concentration on its own does not necessarily reflect the ACTH release. At 4 weeks of age, the ACTH response to stress is more difficult to suppress than in adult animals. This is more obvious after adrenalectomy, where the excessive ACTH secretion was less inhibited by all glucocorticoids used. The time between the last steroid gavage and stress must be considered. In 4-week-old animals the ACTH response 16 h after 12.5 micrograms of dexamethasone was inhibited by 22%, whereas 4 h after the same dexamethasone dose the inhibition was 85%.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor Binding and Biological Activity of IL-α, IL-1β, IL-1β Analogues and an IL-1 Antagonist in A375 Human Melanoma Cells

AbstractA receptor binding assay for IL-1 peptides on human melanoma cells of the A 375 cell line is reported. Strains differing in their sensitivity to the cytotoxic effects of IL-1β were compared. In both strains, binding equilibrium at temperatures between 0° and 37°C was reached after 4 to 8 hours. At 37°C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0°C revealed a single class of high affinity receptors with a similar KD in both IL-1 resistant (0.18±0.07 nM) and sensitive strains (0.14±0.06 nM) but a 10–fold difference in the number of binding sites. Whereas >1000 binding sites per cell were regularly observed in all resistant strains, only 100–200 sites could be detected on the IL-1 sensitive cells. In displacement assays, IL-1β was found to be slightly more potent than IL-1α in both strains. In an attempt to further characteristics the IL-1 binding site in these cells, the binding characteristics and biological activity of 2...

91 URINARY GROWTH HORMONE (GH) – CLINICAL APPLICATION

A radiometric assay for GH was applied to unprocessed urine (technical details previously described). A significant correlation between plasma profiles and urinary GH excretion has been found (i.e. 24 h plasma integrated concentrations (IC) to urine pg/24 h, r = 0.45. Plasma IC 24 h to night urine GH/creatinine 0.69, night plasma IC to night urine 0.58, for all correlations N = 40). In 155 24 h-urines of “normal” children (partly referred for suspected growth problems), a mean GH-excretion of 6.2 ± 6.5 ng/24 h (median 4.1, P. 10 1.3, P. 90 13.6) has been found. No clear relation was found to chronological age, bone age or puberty ratings. 10 patients with “precocious puberty” (5 idiopathic, 5 treated CAH) had a mean excretion of 9.7 ± 5.4 (median 8.4, P. 10 4.6, P. 90 19). In active acromegaly, the values varied from 73 to 500 ng/24 h. Patients with “complete” (N = 9) and “partial” (N = 7) GH-deficiency had a mean (median) excretion of 0.79 (0.75) and 3.3 (1.8) ng/24 h off therapy. The values increased to 6.2 (3.8) and 11.3 (10.5) ng/24 h during therapy (2 IU daily s.c). Before more conclusions can be drawn, the important intraindividual variation of GH-excretion has to be considered. Nevertheless, urinary GH mirrors an actual GH-production over a set time and can be applied to clinical states with suspected “abnormal” GH-production or for therapy survey.