ABSTRACT

The aim of the present study was to investigate how atriopeptin inhibits secretagogue-stimulated ACTH secretion in vitro. Perifused isolated rat anterior pituitary cells were used throughout; the ACTH content of the perifusate was measured by radioimmunoassy.

In the presence of a constant (0·05 nmol/l) concentration of 41-residue corticotrophin-releasing factor (CRF), arginine vasopressin (AVP; 0·05–50 nmol/l) stimulated ACTH secretion in a concentration-dependent manner, the combination of 0·05 nmol CRF/l and 0·5 nmol AVP/l (CRF/AVP) stimulated ACTH release to six- to eightfold above baseline. The effect of CRF/AVP was not modified by tetrodotoxin, but was abolished by CoCl₂ and reduced to about 70% of the control stimulus by nifedipine.

Application of 103–126 residue atriopeptin for 10 min before and 2·5 min during the CRF/AVP stimulus strongly suppressed the evoked release of ACTH, the maximal inhibition was 75–90% at 10 nmol atriopeptin/l. The calcium ionophore ionomycin (200 nmol/l) reversed the effect of atriopeptin while it had no secretagogue activity of its own, and did not enhance the response to CRF/AVP. A variety of blockers of K⁺ channels, 4-amino pyridine, tetraethylammonium, apamine, quinine, but not tolbutamide, effectively antagonized the inhibitory action of atriopeptin (10 nmol/l). None of these drugs altered ACTH release evoked by CRF/AVP. In concentration–response experiments, the half effective concentration of 4-aminopyridine and tetraethyl-ammonium were around 1 mmol/l and 10 nmol/l for apamine. Finally, tetraethylammonium and apamine also antagonized the inhibition of CRF/AVP-evoked ACTH release by 8-Br-cGMP.

These data suggest that (1) at least two types of K⁺ channels, a delayed rectifier and the apamine-sensitive Ca²⁺-activated channel, are functionally important in pituitary corticotroph cells; (2) atriopeptin inhibits CRF/AVP-stimulated ACTH secretion by hyperpolarizing the plasma membrane and thus reducing the uptake of Ca²⁺ into the cells; (3) cGMP is the intracellular mediator of the action of atriopeptin on corticotroph cells.

Journal of Endocrinology (1990) 126, 183–191

ABSTRACT

The current concept of the regulation of adenohypophysial corticotrophin secretion assigns a stimulatory role to neuropeptides of hypothalamic origin and an inhibitory function to adrenocortical glucocorticoid hormones. It has been shown that in isolated perifused rat anterior pituitary cells atriopeptins (1–28) and (5–28), but not (5–25), potently suppress corticotrophin secretion elicited by 41-residue corticotrophin-releasing factor and arginine vasopressin. The action of atriopeptins is associated with a marked increase in the cellular content of cyclic GMP. Moreover, 8-Br-cyclic GMP, a cell membrane permeant analogue of cyclic GMP, also inhibited secretagogue-stimulated corticotrophin release. It is suggested that atriopeptins of hypothalamic and perhaps of cardiac origin may be functionally significant corticotrophin-release inhibiting factors.

Journal of Endocrinology (1990) 125, 39–44

ABSTRACT

The aim of the present study was to characterize the inhibitory action of atriopeptin on secretagogue-evoked ACTH release in vitro.

Perifused isolated rat anterior pituitary cells were exposed to repeated pulses of 41-residue corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP). The net ACTH secretory response to both neurohormones increased progressively with the number of pulses applied, until a maximum hormonal response was reached which was stable for the subsequent period of observation (2–3 h). The maximal secretagogue-evoked hormone release eventually achieved was 4 and 1·7 times greater than the initial response to AVP and CRF-41 respectively. The size of the ACTH response elicited by 50 pmol CRF-41/1 and 500 pmol AVP/1 (CRF/AVP) given together also underwent progressive enhancement. The number of secretagogue pulses required to reach the maximal response to a particular stimulus depended upon the concentration of the secretagogue peptides, higher concentrations favoured a more rapid development of the stable secretory response.

The potency of 103–126 residue atriopeptin to inhibit CRF/AVP-induced ACTH release varied by about 1000-fold depending upon the prior treatment of the cells. In general, cells not previously exposed to secretagogues appeared largely resistant, those under a moderate secretagogue drive were strongly inhibited, and those under intense stimulation were again refractory to inhibition by atriopeptin. In contrast, corticosterone suppressed stimulated ACTH release regardless of the state of the cells.

The data demonstrate that the conditions of cell maintenance are pivotal determinants of the inhibitory effect of atriopeptin on secretagogue-stimulated ACTH release in vitro. The in-vivo correlate of these findings may be that the sensitivity of corticotrophs towards atriopeptin is determined by requirements encoded through the pattern of release of hypothalamic CRF-41 and AVP.

Journal of Endocrinology (1990) 125, 365–373

ABSTRACT

Vasopressin and 41-residue corticotrophin-releasing factor (CRF-41) are physiological mediators of the hypothalamic control of pituitary ACTH secretion, whilst adrenocortical glucocorticoids are the major inhibitory factors regulating ACTH output. In the present study it was investigated in vitro whether the characteristics of early glucocorticoid inhibition of stimulated ACTH secretion would differ depending on the nature of the stimulus and the temporal relationship between secretagogue and steroid.

The experiments were carried out using perifused segments of rat adenohypophysis obtained from randomly cycling female rats. Repeated pulses (5 min) of CRF-41 or vasopressin were given at 1-h intervals for up to 7 h. The net release of ACTH became stable after the second secretagogue pulse. Administration of 0·1 μmol corticosterone/l 30 min before and during a 5-min pulse of 10 nmol CRF-41/l inhibited CRF-41-stimulated ACTH release to 60% of control. Stimulated hormone release remained suppressed at 90 min after the start of the corticosterone infusion and returned to control levels by 150 min. If corticosterone treatment (35 min total exposure) was started simultaneously with the CRF-41 pulse, no inhibitory effect of the steroid was observed at any subsequent time-point examined (60,90,120 and 150 min). In contrast, vasopressin-stimulated ACTH release was inhibited by approximately 50% when corticosterone was applied before, or simultaneously with, a 5-min pulse of 10 nmol vasopressin/l. The synthetic glucocorticoid type II receptor agonist RU28362, administered 30 min before and during a 5-min pulse of 10 nmol CRF-41/l, reduced CRF-41-stimulated ACTH release to 50% of control up to 2·5 h after the start of RU28362 application (although inhibition after 35 min exposure was not statistically significant). Inhibition of ACTH release stimulated by 10 nmol vasopressin/l was observed within 35 min of steroid application and was maintained up to 2·5 h after the initial application of RU28362. The action of RU28362 on CRF-41-stimulated ACTH release was blocked by inhibitors of transcription (actinomycin D) and translation (puromycin); notably these drugs did not modify the ACTH response to CRF-41. In contrast, actinomycin D as well as puromycin reduced vasopressin-stimulated ACTH release.

The data suggest that: (1) the timing of steroid application is important in determining the early glucocorticoid inhibition of CRF-41- but not vasopressin-stimulated ACTH secretion; (2) CRF-41 and vasopressin mobilize different pools of ACTH from the anterior pituitary gland; (3) type II glucocorticoid receptors and synthesis of new protein(s) are involved in the early inhibitory action of glucocorticoids; (4) depending on the timing and nature of the incident secretagogue, differential negative feedback inhibition of ACTH secretion may occur at the pituitary level in vivo.

Journal of Endocrinology (1991) 129, 261–268

ABSTRACT

Oxytocin may function as a hypothalamic releasing hormone for prolactin and ACTH secretion in the rat. In the present study we have investigated the properties of putative oxytocin receptors in the rat adenohypophysis by radioligand-binding assay.

A novel oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid),2-(ortho-methyl)-Tyr²-Thr⁴-Orn⁸-Tyr⁹-NH₂]-vasotocin (OTA) was radioiodinated by the iodogen method to a specific activity of 0·6 nCi/fmol. The radioiodinated derivative ¹²⁵I-labelled OTA (¹²⁵I-OTA) was reacted with membrane suspensions prepared from the uterus or adenohypophysis of female rats which were (a) ovariectomized for 7 days, (b) ovariectomized and treated with 5 μg oestradiol-17β 48 h before death or (c) implanted with a piece of silicone elastomer tubing containing 50 mg diethylstilboestrol (DES) 5 days before death. In uterine as well as the pituitary membrane suspensions, the radioligand was bound reversibly and with high affinity (dissociation constants 0·2 ± 0·1 and 0·1±0·01 nmol/l respectively; means + s.e.m., n=3) to a single class of sites with limited binding capacity, which varied with the type of pretreatment. Oestradiol-17β increased the binding capacity fivefold in the uterus in ovariectomized rats, but only very low specific radioligand binding was found in pituitary preparations from the same animals. Treatment with DES markedly increased the number of receptors in both the uterus and the adenohypophysis. Studies with several agonist and antagonist analogues revealed no difference in the ligand specificity of the uterine and adenohypophysial sites binding ¹²⁵I-OTA, indicating that they are the same species of receptor. Furthermore, ligand-binding studies, carried out with tritiated vasopressin as tracer in pituitary membrane preparations, showed that OTA is not bound by pituitary vasopressin receptors at concentrations below 1 μmol/l.

In summary, ¹²⁵I-OTA is a highly specific radioligand suitable for the analysis of pituitary oxytocin receptors. The low number of oxytocin receptors in the adenohypophysis of rats treated with oestradiol-17β suggests that the prolactin-stimulating action of oxytocin is mediated by highly efficient transmembrane signalling.

Journal of Endocrinology (1989) 122, 465–470

The role of prostaglandins in the control of aldosterone production was studied in isolated rat glomerulosa cells. Exogenous prostaglandin E2 in concentrations above 10⁻⁹ mol/l increased the production rate of aldosterone; this effect was attenuated by the competitive antagonist, 7-oxa-13-prostynoic acid. Prostaglandin F_2α (10⁻⁹–10⁻⁵ mol/l) failed to influence the production rate of aldosterone. The aldosterone-stimulating effect of the prostaglandin precursor, arachidonic acid (5 × 10⁻⁴ mol/l), could not be blocked by inhibitors of prostaglandin synthesis. Basal production rate of aldosterone was not significantly influenced by non-steroidal anti-inflammatory drugs. Glomerulosa cells were stimulated by angiotensin II; this effect was not potentiated by arachidonic acid and was reduced only slightly by indomethacin. The cells were also stimulated by corticotrophin and potassium ions. The effect of these substances was not potentiated by arachidonic acid and was not inhibited by non-steroidal anti-inflammatory drugs. These results do not confirm the presumption that intra-adrenal prostaglandins play an essential role in the control of aldosterone secretion. Some effects of arachidonic acid and its antagonist, eicosatetraynoic acid, on aldosterone production are considered to be independent of changes in prostaglandin synthesis.

ABSTRACT

Previous data show that corticotrophin-releasing factor-41 (CRF-41), arginine vasopressin (AVP) and oxytocin are released into hypophysial portal blood. It has been presumed that the CRF-41 originates mainly from parvicellular neurones of the paraventricular nuclei (PVN); however, AVP and oxytocin could also be derived as a consequence of preterminal release from magnocellular projections to the neurohypophysis. The latter has been suggested to be the case for AVP as assessed by studies of the median eminence in vitro. Here we have investigated the source of CRF-41, AVP and oxytocin in hypophysial portal blood of adult male Wistar rats 8–10 days after surgical lesioning of the PVN. In PVN-lesioned animals the output of CRF-41 into hypophysial portal blood was reduced by about 90%, and that of oxytocin by about 40%: however, the output of AVP into portal blood was reduced only by about 10%. The release of AVP into portal blood increased after adrenalectomy; this increased release could be returned to normal by treatment with dexamethasone. No change of AVP release occurred after adrenalectomy in animals in which the PVN had been lesioned. These results show (i) that most of the CRF-41 released into hypophysial portal blood is derived from the PVN, (ii) that in PVN-lesioned animals AVP and oxytocin release remains at near normal or 60% of normal respectively, suggesting that a substantial amount of both neuropeptides in portal blood is derived as a consequence of preterminal release from supraoptic nuclei projections in the median eminence, and (iii) that glucocorticoid feedback inhibition of AVP release is exerted at the level of the PVN.

Journal of Endocrinology (1990) 125, 175–183

Neurotoxic lesions of the arcuate nucleus by neonatal treatment with monosodium glutamate (MSG) resulted in a decrease of plasma GH levels without affecting plasma corticosterone concentration. The corticoliberin activity of the pituitary stalk median eminence (SME) in MSG-treated animals was similar to that of litter-mate controls. Growth hormone releasing (somatoliberin) activity of the SME, tested after placing an anterolateral cut around the medial basal hypothalamus, was significantly lower in MSG-treated than in control animals. It was concluded that a substantial proportion of the somatoliberin neurones are found in the arcuate nucleus.

The possible role of the neural connections of the medial-basal hypothalamus (MBH) in the maintenance of GH releasing activity of the pituitary stalk median eminence (SME) was investigated. Male rats, subjected to sham-operation and to complete and anterolateral cuts around the MBH were used 7–8 days after surgery. Electrical stimulation of neural structures within the MBH caused an increase of plasma GH in pentobarbitone- as well as in urethane-anaesthetized animals. In sham-operated rats the rise of plasma GH levels was apparent only after completion of 10 min of electrical stimulation, while in animals with complete or anterolateral cuts an increase was already evident during electrical stimulation. The results suggest that depolarization of somatostatin secreting fibres in the median eminence may be responsible for the delay in the rise of GH levels in sham-operated rats, while the increment can be attributed to a GH releasing principle in the hypothalamus. Acidic extracts of the SME of rats with complete or anterolateral cuts stimulated the release of GH by primary cultures of rat anterior pituitary cells. Microinjection of 0·05 SME equivalents of SME extract into the anterior pituitary gland of urethane-anaesthetized rats produced a rise in plasma GH levels within 3 min of injection. These data favour the existence of a GH releasing factor, and suggest that the ventromedial and arcuate hypothalamic nuclei are major sites of production of this releasing hormone.

The role of nerve cells of the arcuate nucleus and endogenous opioid peptides in the regulation of GH and prolactin secretion has been investigated. Electrical stimulation of the medial-basal hypothalamus (MBH) for 10 min raised plasma levels of both hormones in male rats anaesthetized with pentobarbitone sodium. Plasma hormone levels increased within 5 min after the termination of the stimulus, while no marked changes were found during stimulation. The GH response to the electrical stimulus was substantially reduced in rats with arcuate lesions induced by neonatal treatment with monosodium-l-glutamate (MSG). By contrast, the size of the prolactin response was not altered by MSG treatment. The opiate receptor antagonist naloxone (10 mg/kg, i.v.) failed to influence GH secretion induced by electrical stimulation in either control or MSG-treated animals. The post-stimulus rise of plasma prolactin levels was attenuated by naloxone in control rats, while the same dose of the drug was ineffective in rats which had been exposed to MSG. We conclude that endogenous opioids participate in the increase of prolactin release upon electrical stimulation of the MBH but are not involved in the GH secretory response. Arcuate neurones are important in the maintenance of the GH response to electrical stimulation. By contrast, lesioning of the arcuate nucleus failed to affect the prolactin secretory response elicited by MBH stimulation. However, prolactin release in MSG-treated rats appeared less susceptible to the inhibitory action of naloxone, suggesting a possible supersensitivity towards endogenous opioids.