Comparative Chromatography of Hypothalamic Corticotrophin-Releasing Factors

Gillies, Glenda; Puri, A.; Linton, Elizabeth A.; Lowry, P. J.

doi:10.1159/000123860

Cited by 26 publications

(9 citation statements)

References 28 publications

(42 reference statements)

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“…In conclusion, we have demonstrated a development of CRF, AVP and SRIF production by neurons over extended periods in culture as assessed by their peptide content and increasing responses to depolarizing stimuli. Such a system should prove useful in the study of the effects of environmental factors such as steroids, peptides and neurotransmitters on the maturation of peptidergic neurons and the synthesis and release of CRF, AVP and SRIF.Past work on the nature of corticotropin-releasing factor (CRF) in hypothalamic extracts has led to the conclusion that it is not a single, simple peptide, but a variety of factors which interact and synergise together [ 13], Two of these fac tors are the 41 amino acid CRF [34] and vasopressin (AVP) [11,13,28]. The many mechanisms operating in vivo to achieve the homeostasis of the hypothalamo-pituitaryadrenal axis complicate the study of CRF release.…”

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confidence: 99%

Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

1987

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The concomitant release of corticotropin-releasing factor (CRF), vasopressin (AVP) and somatostatin (SRIF) has been followed from primary cultures of rat hypothalamic neurons. 18-day-old fetal rat hypothalami were dissociated enzymatically and mechanically, then plated and maintained in a serum-containing medium at a density of 2.5 × 10⁶ cells per dish (equivalent to 3 hypothalami). Cultured neurons remained viable for up to 6 weeks, and peptide release was followed by immuno-assay between days 14 and 39 in culture. The incubation media were concentrated on C₄ and C₈ silica columns to facilitate detection of CRF and AVP. Peptide release was measured at various times up to 4 h, at which point it was still increasing. To optimise measurements, taking into account peptide degradation, a 1-hour incubation period was chosen for further studies. Release of CRF, AVP and SRIF by 56 mM K⁺ or 10 µM veratridine was statistically significantly greater than basal (p < 0.01) and was Ca²⁺-dependent. For CRF and AVP, stimulated release increased considerably with the age of culture, whereas SRIF release was steadier. Basal release for all 3 peptides did not fluctuate greatly over this period. Basal and stimulated release of the peptides continued over at least 5 successive 1-hour periods. At day 35 of culture, the peptide content was still increasing in a pattern which paralleled the increasing content in hypothalami freshly removed from age-matched rats. In conclusion, we have demonstrated a development of CRF, AVP and SRIF production by neurons over extended periods in culture as assessed by their peptide content and increasing responses to depolarizing stimuli. Such a system should prove useful in the study of the effects of environmental factors such as steroids, peptides and neurotransmitters on the maturation of peptidergic neurons and the synthesis and release of CRF, AVP and SRIF.

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confidence: 99%

Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

1987

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“…How¬ ever, the antibody-quenching experiments showed that immunoneutralization of AVP caused a 60% reduction in bioactivity (Fig. 2), which is indicative of synergism of VP with other factors in the CRF complex (Gillies & Lowry, 1979Gillies et al , 1984. As log dose-response lines to stalk median eminence extract, oCRF-41 and AVP are non-parallel it is incorrect to compare potencies, but the above results suggest a significant difference in the activities of extracted and released ACTH-releasing activity.…”

Section: Discussionmentioning

confidence: 96%

“…A rat 41 amino acid CRF with •One stalk median eminence (SME) extract = 3-4mg tissue homogenized lOmM-HCl containing 1 g ascorbic acid/I . tValues collected over a number of years Gillies et al , 1984present results).…”

Section: Discussionmentioning

confidence: 99%

“…Vasopressin appears to play a role in controlling ACTH release in vitro (Gillies & Lowry, 1979Beny & Baertschi, 1981; Buckingham, 19826;Sokol & Zimmerman, 1982; Gillies, Puri, Linton & Lowry, 1984) and in vivo (Rivier & Vale, 1983a) and synergism between AVP and oCRF-41 has been amply demon¬ strated in vivo (Liu, Muse, Contreras et al 1983;Rivier & Vale, 1983 , b) as well as in vitro (Gillies, Linton & Lowry, 1982;Turkelson, Thomas, Arimura et al 1982). We wished to investigate the nature of released, rather than extracted ACTH-releasing activity and adopted a system of superfusion of hypothalamic pieces.…”

Section: Discussionmentioning

confidence: 99%

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Involvement of rat corticotrophin-releasing factor-41-related peptide and vasopressin in adrenocorticotrophin-releasing activity from superfused rat hypothalami in vitro

Gillies¹,

Puri²,

Hodgkinson³

et al. 1984

Journal of Endocrinology

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Superfused rat hypothalamic pieces stimulated with a high K+ medium released corticotrophin-releasing factor (CRF) as detected in the rat isolated anterior pituitary cell column bioassay. This activity has components related to ovine CRF-41 (oCRF-41) and vasopressin as assessed by the effects of specific antisera on the ACTH-releasing activity of the hypothalamic column effluent. Release of immunoactive vasopressin paralleled that of CRF bioactivity. Immunoactive oCRF-41 could not be detected, as it is likely to be below the sensitivity of present radioimmunoassays.

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“…Acute organ, slice or synaptosomal preparations may suffer from cell damage, poor diffusion and the presence of sequestered neuro¬ transmitters from extrahypothalamic cell bodies. Therefore, in order to study the control of the hypothalamic corticotrophin-releasing factor (CRF) com¬ plex (Gillies, Linton & Lowry, 1982;Gillies, Puri, Linton & Lowry, 1984) we have established a system of long-term primary culture of fetal rat hypothalamic neurones (Clarke, Lowry & Gillies, 1987) coupled with the direct measurement of the released 41 amino acid-containing CRF (CRF-41) and vasopressin (AVP) by immunoassay. As with most studies, these earlier cultures were maintained in a traditional medium supplemented with serum which provides the hormones, nutrients, binding proteins and attachment factors necessary for the survival and growth of cells in vitro.…”

Section: Introductionmentioning

confidence: 99%

Comparison of peptide release from fetal rat hypothalamic neurones cultured in defined media and serum-containing media

Clarke

Gillies²

1988

Journal of Endocrinology

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Primary cultures of rat hypothalamic neurones were maintained either in a serum-supplemented medium or in a serum-free chemically defined medium for up to 6 weeks. The release of the 41 amino acid-containing peptide, corticotrophin-releasing factor (CRF-41), vasopressin (AVP) and somatostatin (SRIF) were followed using immunoassays. In response to K+ (56 mmol/l) depolarization both the quantities of peptides released and the magnitude of responses were significantly greater from cultures maintained in the fully supplemented defined medium. As a consequence, release of CRF-41 and AVP could be measured directly, without requiring the concentration step necessary for cultures grown in serum. The response to K+ depolarization increased with the age of the culture, suggesting neuronal maturation. Responses to K+ depolarization were Ca2+-dependent, and the addition of corticosterone (100 nmol/l) to the defined medium caused a significant reduction in the response of neurones secreting CRF-41 and AVP, but not those secreting SRIF, to depolarization. This suggests the retention in vitro of the responsiveness of stress-associated neuropeptides to the negative feedback effects of corticosterone. Neurones producing CRF-41 and AVP responded significantly in a dose-dependent manner to acetylcholine stimulation, whereas those producing SRIF did not. As cultures matured, the CRF-41- and AVP-producing neurones became more sensitive to acetylcholine with the maximal response at 1 nmol acetylcholine/l. In conclusion, the culture of rat hypothalamic neurones is improved in terms of peptide output when the cultures are maintained in a defined medium.(ABSTRACT TRUNCATED AT 250 WORDS)

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Comparative Chromatography of Hypothalamic Corticotrophin-Releasing Factors

Cited by 26 publications

References 28 publications

Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

Involvement of rat corticotrophin-releasing factor-41-related peptide and vasopressin in adrenocorticotrophin-releasing activity from superfused rat hypothalami in vitro

Comparison of peptide release from fetal rat hypothalamic neurones cultured in defined media and serum-containing media

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