Glenda Gillies

Interleukin-1 Directly Stimulates the Release of Corticotrophin Releasing Factor from Rat Hypothalamus

While interleukin-1 (IL-1), a monocyte-derived polypeptide, clearly stimulates the hypothalamo-pituitary-adrenal (HPA) axis, its precise site of action is controversial. In these studies, the possibility of a hypothalamic and/or a pituitary site of action was investigated in vitro, using incubated rat hypothalami and perifused dispersed pituitary cells. Both forms of IL-1, IL-1 alpha and IL-1 beta, produced a dose-dependent stimulation of CRF-41 release from incubated rat hypothalami in the dose range of 1-100 U/ml (p less than 0.01). However, concentrations of both interleukins of 1-1,000 U/ml given as 10-min infusions had no effect on ACTH release from dispersed pituitary cells. Moreover, IL-1 beta, used in the concentration range of 1-100 U/ml, was unable to potentiate CRF-41-induced ACTH release. These data therefore provide evidence that at least the acute stimulatory effects of IL-1 on the HPA axis are predominantly mediated via a direct stimulation of hypothalamic CRF-41, suggesting that the hypothalamus may provide an interface between the neuroendocrine and immune axes.

ANGIOTENSIN II STIMULATES THE RELEASE OF ACTH FROM DISPERSED RAT ANTERIOR PITUITARY CELLS

In a rat anterior pituitary dispersed cell system, angiotensin II was found to stimulate the release of ACTH at concentrations ranging from 100 pmol/l to l μmol/l, with a maximal response being given by 10 nmol/l. The angiotensin II antagonist, saralasin, was able to block completely at a concentration of l μmol/l the stimulation of ACTH induced by 10 nmol/l angiotensin II, but had no effect on the basal release. The log dose‐response curve for ACTH release by angiotensin II was shifted to the right in a parallel fashion by saralasin 10 nmol/l, suggesting competitive antagonism. The stimulation of ACTH release by a rat stalk‐median eminence extract or by arginine vasopressin was unaffected by saralasin. The data are interpreted as suggesting that rat corticotrophs contain angiotensin II receptors, and that these may be involved in ACTH release in response to hypovolaemic stress.

Pro-opiocortin related peptides in human pituitary and ectopic ACTH secreting tumours.

Basal and stimulated secretion of N‐terminal pro‐opiocortin (Pro‐γ‐MSH), ACTH and LPH from seven pituitary and three ectopic ACTH secreting tumours have been studied in vitro using a perfused isolated cell system. The peptides were shown to be released concomitantly and in equimolar amounts. The pituitary tumours responded to stimulation with rat stalk median eminence extracts (SME) and synthetic AVP. However, peptide release from the ectopic tumours, although pulsatile, remained autonomous. Prior to surgery, gel‐chro‐matographic profiles of plasma immunoreactive ACTH showed only one peak, which eluted in the position of 1–39 ACTH, in patients with the pituitary tumours, but there was a second peak of large molecular weight ACTH present in the plasma from those with the ectopic ACTH syndrome. This second form of ACTH could not be detected in any of the tumour cell column effluents. An eighth pituitary tumour was atypical, in its unusually large size, clinically aggressive nature and spectrum of peptide release. Although peptide release in response to stimulation with SME was similar to that observed with the other pituitary tumours, the chromatography of the plasma ACTH resembled the ectopic plasma pattern, showing two peaks of immunoreactivity.

The CRFs and their control: Chemistry, physiology and clinical implications

The 41-amino acid CRF fulfils all the criteria for a corticotrophin releasing factor, although considerable evidence suggests that other factors, particularly VP, also play a physiologically significant role in controlling ACTH release. Although human CRF has now been identified as a 41-residue peptide, most studies to date have used oCRF-41 in their exploration of the physiology and pathology of the hypothalamic--pituitary--adrenal axis. Low doses of oCRF-41 appear to be safe, and for specific tests of the readily-releasable pool of ACTH and related peptides 100 micrograms is a practical dose for most purposes. Although serious side-effects have only been noted at doses above 100 micrograms, it is reasonable to monitor all patients administered CRF-41 with great care, and in particular to be alert to hypotension, especially in patients with corticosteroid deficiency. There is little doubt that, in combination with the standard insulin-tolerance test, the CRF test is a useful means of diagnosing hypothalamic or portal dysfunction in patients with secondary adrenal failure. However, in the diagnosis and differential diagnosis of Cushings syndrome, the role of the CRF test remains unclear. In normal subjects, a high basal cortisol level usually inhibits the response to CRF, such that a greatly enhanced response is suggestive of pituitary-dependent Cushings syndrome. In patients with diagnosed ACTH-dependent Cushings syndrome, an absent response to CRF predisposes towards an ectopic source of ACTH. However, there are exceptions in all directions, and it is uncertain whether the CRF test will prove of greater value than the traditional procedures, such as the dexamethasone suppression test. The differential diagnosis of depression and Cushings disease may be its greatest value. In terms of treatment, there are as yet few data on the usefulness of CRF in expediting recovery of the pituitary-adrenal axis following long-term suppression, such as in patients with Cushings syndrome treated by removal of a unilateral adenoma or trans-sphenoidal microadenomectomy. It is possible that such treatment may eventually be a useful application of CRF, although data are not yet available.

SECRETION OF ACTH, LPH AND β‐ENDORPHIN FROM HUMAN PITUITARY TUMOURS IN VITRO

Basal and stimulated secretion of immunoreactive ACTH, LPH and β‐endorphin from four human pituitary tumours has been studied in vitro using a superfused, isolated cell system. Chromatography of cell secretions under acid‐dissociating conditions demonstrated that the human tumour cells released immunoreactive peptides with the elution profiles of αh (1–39) ACTH, βh‐LPH, γh‐LPH and βh‐endorphin confirming that βh‐endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from βh‐LPH in blood. No α‐ or βh‐MSH, nor any higher molecular weight forms of ACTH or LPH were detected. The cells from all four tumours responded to stimulation with rat stalk‐median eminence extract (SME) and synthetic AVP with a concomitant release of ACTH, β‐LPH, γ‐LPH and γ‐endorphin. In contrast to the isolated rat anterior pituitary cells, the pattern of responses to SME and AVP were indistinguishable and the release provoked by rat SME could be accounted for virtually entirely by its vasopressin content. No stimulation of release was observed when the cells were exposed to a variety of biogenic amines. Addition of hydrocortisone to the perfusion buffer of two tumours resulted in a slow inhibition of both basal and stimulated ACTH and LPH release. These data demonstrate that human pituitary tumour tissue from patients with Cushings disease and Nelsons syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.

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

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 x 10(6) 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 microM veratridine was statistically significantly greater than basal (p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)