ACTH release in pituitary cell cultures. Effect of neurogenic peptides and neurotransmitter substances on ACTH release induced by hypothalamic corticotropin releasing factor (CRF).

The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HY-CRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0.0165 HE/ml and 0.5 HE/ml. Relative high doses of lysine-vasopressin showed a slight additive effect on the release of ACTH induced by 0.1 HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrotropin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, beta-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and gamma-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.     

In vivo and in vitro release of ACTH by synthetic CRF

The 41-residue corticotropin releasing factor (CRF) was synthesized by the solid phase method. The synthetic CRF and arginine vasopressin (AVP) were examined for ACTH releasing activity and effects on the release of 5 other pituitary hormones in vivo and in vitro. Injection of the CRF into pharmacologically blocked rats increased plasma corticosterone levels in a dose-related manner. The minimum effective dose was 1.6 x 10(-12) mol/100 g body weight. CRF also significantly stimulated release of ACTH-like immunoreactivity in a dose-related manner from rat pituitary quarters beginning at a concentration of 10(-9) M. AVP, a peptide known to have CRF activity, exhibited slightly lower corticotropin releasing activity than the CRF at equimolar dose levels. Secretion of other pituitary hormones was not appreciably altered by either the CRF or AVP.     

Comparison in male and female rats of ACTH content and response to corticotrophin-releasing factor (CRF) of cultured adenohypophyseal cells and in vivo hypothalamic CRF content.

Although mean ACTH concentration was significantly higher in cultured adenohypophyseal cells from female than from male rats, there was no significant sex difference in the ACTH secretory response of these cells to hypothalamic extract containing CRF. Hypothalamic CRF content in the “basal” state $\text{in}$$\text{vivo}$ was slightly higher in female than $\text{in}$ male rats.     

Inhibition of spontaneous ACTH release and improved response to CRF in sheep premedicated with the enkephalin analogue DAMME

The effect of IV injections of an enkephalin analogue (DAMME) in sheep prior to the administration of CRF has been compared with chlorpromazine-morphine (CPZ-M) injections as well as a chlorpromazine-morphine-nembutal (CPZ-M-N) regime. The results showed that the administration of DAMME 60 minutes prior to CRF injections, gave more consistent and more sustained inhibition of spontaneous plasma ACTH and plasma cortisol secretion than either CPZ-M or CPZ-M-N pre-treatment. Changes in plasma ACTH and plasma cortisol levels were more readily detectable in DAMME-treated sheep as compared with saline controls when 50 micrograms injections of CRF were given into the carotid artery. This suggests that DAMME may inhibit endogenous CRF release by an action at hypothalamic level or above.     

Attenuation of corticotropin releasing factor-induced hypotension in anesthetized rats with the CRF antagonist, alpha-helical CRF9-41; comparison with effect on ACTH release.

The effect of pretreatment with the corticotropin releasing factor (CRF-41) antagonist, alpha-helical CRF(9-41), on the hypotensive response obtained on peripheral administration of CRF-41 has been assessed in anesthetized Wistar rats. A single IV bolus dose of rat CRF-41 (2 nmol, at 0 min) produced a hypotensive effect which was rapid in onset (-52 mmHg at +1 min) and sustained throughout the 60-min study period (-42, -40, -26 and -16 mmHg at +3, +10, +30 and +60 min, respectively). The antagonist [alpha CRF(9-41)] was administered in consecutive bolus doses of 12.5, 25 and 50 nmol at -15, -10 and -5 min, respectively. This had no effect on mean arterial blood pressure (MABP) or heart rate, nor did it change significantly the magnitude of the initial rapid fall in MABP when CRF-41 was administered (-45 mmHg at +1 min). However, following pretreatment with alpha CRF(9-41), MABP returned to control values within 3 min and the sustained period of hypotension was completely blocked. Administration of CRF-41 resulted in 44% and 142% increases in norepinephrine and epinephrine measured at +60 min. Pretreatment with the antagonist attenuated the rise in circulating catecholamine levels observed after CRF-41 administration. In comparison, pretreatment with the antagonist did not alter the ACTH response to CRF-41 at +1 and +3 min and only reduced ACTH levels by 28% (p less than 0.05), 43% (p less than 0.001) and 41% (p less than 0.01) at 10, 30 and 60 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Footshock-induced changes in brain catecholamines and indoleamines are not mediated by CRF or ACTH

Stressful treatments have long been associated with increased activity of brain catecholaminergic and serotonergic neurons. An intracerebroventricular (icv) injection of the corticotropin-releasing factor (CRF) also activates brain catecholaminergic neurons. Because brain CRF-containing neurons appear to be activated during stress, it is possible that CRF mediates the catecholaminergic activation. This hypothesis has been tested by assessing the responses in brain catecholamines and indoleamines to footshock in mice pretreated icv with a CRF receptor antagonist, and in mice lacking the gene for CRF (CRFko mice). Consistent with earlier results, icv administration of CRF increased catabolites of dopamine and norepinephrine, but failed to alter tryptophan concentrations or serotonin catabolism. A brief period of footshock increased plasma corticosterone and the concentrations of tryptophan and the catabolites of dopamine, norepinephrine and serotonin in several brain regions. Mice injected icv with 25 microg alpha-helical CRF(9-41) prior to footshock had neurochemical responses that were indistinguishable from controls injected with vehicle, while the increase in plasma corticosterone was slightly attenuated in some experiments. CRFko mice exhibited neurochemical responses to footshock that were indistinguishable from wild-type mice. However, whereas wild-type mice showed the expected increase in plasma corticosterone, there was no such increase in CRFko mice. Similarly, hypophysectomized mice also showed normal neurochemical responses to footshock, but no increase in plasma corticosterone. Hypophysectomy itself elevated brain tryptophan and catecholamine and serotonin metabolism. Treatment with ACTH icv or peripherally failed to induce any changes in cerebral catecholamines and indoleamines. These results suggest that CRF and its receptors, and ACTH and other pituitary hormones, are not involved in the catecholamine and serotonin responses to a brief period of footshock.     

Inhibition of CRF- and urotensin I-stimulated ACTH release from goldfish pituitary cell columns by the CRF analogue alpha-helical CRF-(9-41)

alpha-Helical CRF-(9-41) is an analogue of corticotropin-releasing factor (CRF) that antagonizes CRF-stimulated ACTH release in rats. In the present study, alpha-helical CRF-(9-41) was tested to determine whether it would antagonize the ACTH-releasing activity of CRF or urotensin I (UI) observed with superfused, dispersed goldfish anterior pituitary cells. At a concentration of 4 microM, alpha-helical CRF-(9-41) completely blocked the ACTH-releasing activity of 100 nM CRF or 100 nM UI. The inhibitor by itself showed little intrinsic ACTH-releasing activity. This investigation reveals similarities in the CRF-antagonism of alpha-helical CRF-(9-41) in the teleost and mammalian pituitary in vitro. It also provides are similar and suggests that alpha-helical CRF-(9-41) may be useful as a tool to investigate the effects of CRF-like and UI-like peptides in teleost fishes.     

Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system

Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G-100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10-min exposure to these secretagogues. More than 80% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF.     

Actin polymerization overshoots and ATP hydrolysis as assayed by pyrene fluorescence

We investigate via stochastic simulation the overshoots observed in the fluorescence intensity of pyrene-labeled actin during rapid polymerization. We show that previous assumptions about pyrene intensity that ignore the intensity differences between subunits in different ATP hydrolysis states are not consistent with experimental data. This strong sensitivity of intensity to hydrolysis state implies that a measured pyrene intensity curve does not immediately reveal the true polymerization kinetics. We show that there is an optimal range of hydrolysis and phosphate release rate combinations simultaneously consistent with measured polymerization data from previously published severing and Arp2/3 complex-induced branching experiments. Within this range, we find that the pyrene intensity curves are described very accurately by the following average relative intensity coefficients: 0.37 for F-ATP actin; 0.55 for F-ADP + P_i actin; and 0.75 for F-ADP actin. Finally, we present an analytic formula, which properly accounts for the sensitivity of the pyrene assay to hydrolysis state, for estimation of the concentration of free barbed ends from pyrene intensity curves.     

Effects of bromocriptine and cyproheptadine on basal and corticotropin-releasing factor (CRF)-induced ACTH release in a patient with Nelson's syndrome

The effects of bromocriptine, a dopamine agonist, and cyproheptadine, a serotonin antagonist, on basal and corticotropin-releasing factor (CRF)-stimulated ACTH release were investigated in a 40-year-old female patient with Nelson's syndrome. Oral administration of either bromocriptine (2.5 mg) or cyproheptadine (8 mg) caused a marked drop in plasma ACTH levels. Intravenous administration of synthetic ovine (o) CRF (50 micrograms) produced an exaggerated response of plasma ACTH. Short-term (3-week) treatment with either bromocriptine (7.5 mg/day) or cyproheptadine (12 mg/day) resulted in a marked suppression of basal ACTH release. Furthermore, a blunted response of plasma ACTH to oCRF was observed after short-treatment with either drug. However, after a longer period of treatment with cyproheptadine (18-week), plasma ACTH levels rose again and hyperresponsiveness to oCRF was restored to the pretreatment levels. These data indicate that synthetic oCRF is a potent secretagogue for ACTH release in a patient with Nelson's syndrome. It is suggested that bromocriptine and cyproheptadine are effective drugs in reducing basal and CRF-stimulated ACTH release, possibly acting at the pituitary level in this case. However, the apparent refractoriness after chronic treatment with cyproheptadine may limit its therapeutic use in the present case.     

Chromophore release from kraft pulp by purified streptomyces roseiscleroticus xylanases

Treating kraft pulps with the crude xylanase from Streptomyces roseiscleroticus followed by alkali extraction reduces the kappa number in a linear manner with enzyme doses up to about 3 IU/gm of oven-dry pulp. The enzyme complex consists of four isoenzymes designated Xyl1, Xyl2, Xyl3 and Xyl4. Each can release chromophores when used alone and each can facilitate alkali extraction to reduce the kappa number, but their relative abilities are different. Of the four isozymes, Xyl4 releases the least color and 237-nm-absorbing material whereas Xyl3 releases the most. Xyl4 best enhances the ability of alkali to reduce the kappa number. The UV absorption spectrum of the material released by alkali extraction differs significantly from the spectral characteristics of that released during enzyme treatment. The alkali-solubilized material has a maximum absorptivity at 265 nm and relatively little absorptivity at 237 nm. The material released during enzyme treatment absorbs strongly at 205 and 237 nm. UV/VIS spectroscopy of the enzyme- or alkali-released material does not show a characteristics lignin peak at 280 nm, nor does it reveal any notable peaks in the visible region. Analysis of the material released by enzyme treatment revealed more than 40 product peaks after fractionation by reversed-phase HPLC. We observed many products with strong UV absorption. These were relatively hydrophilic. Fewer products absorbed in the visible region. These were more hydrophobic. All four isoenzymes exhibit endo-action patterns; none forms xylose from oat-spelt xylan. The action patterns fell into two groups: endo-1 enzymes (Xyl1 and Xyl3) formed xylotriose (X₃) and other lower oligosaccharides as the predominant products; endo-2 enzymes (Xyl2 and Xyl4) formed roughly equimolar amounts of X₃, xylotetraose (X₄), and xylopentaose (X₅), and tended to leave larger amounts of undigested higher oligosaccharides.     

The release of growth arrest by microinjection of adenovirus E1A DNA.

The induction of DNA synthesis in growth-arrested mouse fibroblasts (NIH 3T3) was studied by microinjection of different constructs of adenovirus DNA using SV40 DNA and plasmid DNA as positive and negative controls. The E1A region of adenovirus types 2 and 12 appears to be sufficient to induce cellular DNA synthesis after growth arrest in approximately 30% of the cells and both 13S and 12S cDNA constructs mediate this effect. The presence of the E1A protein products as assayed by immunofluorescence does not strictly correlate with the induction of DNA synthesis in microinjected cells in contrast to the SV40 large T-antigen. Microinjection of truncated fragments of the Ad12 E1A region suggests, however, that the protein products of 12S and 13S may be involved in the induction process. A sequence comparison of the SV40 T-antigen and the adenovirus E1A products identified a region of significant homology providing a basis for a hypothesis concerning the evolution of T-antigen genes in DNA viruses.