Outcome of Endoscopic Transsphenoidal Surgery in Combination with Somatostatin Analogues in Patients with Growth Hormone Producing Pituitary Adenoma

Objective

To determine the efficacy of endoscopic surgery in combination with long-acting somatostatin analogues (SSAs) in treating patients with growth hormone (GH)-secreting pituitary tumor.

Methods

We performed retrospective analysis of 133 patients with GH producing pituitary adenoma who underwent pure endoscopic transsphenoidal surgery in our center from January 2007 to July 2012. Patients were followed up for a range of 3-48 months. The radiological remission, biochemical remission and complication were evaluated.

Results

A total of 110 (82.7%) patients achieved radiological complete resection, 11 (8.2%) subtotal resection, and 12 (9.0%) partial resection. Eighty-eight (66.2%) patients showed nadir GH level less than 1 ng/mL after oral glucose administration. No mortality or severe disability was observed during follow up. Preoperative long-acting SSA successfully improved left ventricle ejection fraction (LVEF) and blood glucose in three patients who subsequently underwent success operation. Long-acting SSA (20 mg every 30 days) achieved biochemical remission in 19 out 23 (82.6%) patients who showed persistent high GH level after surgery.

Conclusion

Endoscopic transsphenoidal surgery can biochemically cure the majority of GH producing pituitary adenoma. Post-operative use of SSA can improve biochemical remission.     

垂体瘤转化基因在垂体大腺瘤中表达的研究

目的 探讨垂体瘤转化基因（PTTG）在垂体大腺瘤中的表达及意义。方法收集经手术和病理证实的垂体大腺瘤患者40例，其中无功能腺瘤22例，生长激素（GH）腺瘤8例，泌乳素（PRL）腺瘤10例。同期经手术和病理证实的垂体微腺瘤11例为对照组，其中促肾上腺皮质激素（ACTH）腺瘤8例。PRL腺瘤3例。采用免疫组化技术（LSAB法）检测垂体大腺瘤和垂体微腺瘤中PTTG的表达水平。结合临床资料及影像学分级标准，分析PTTG表达水平与垂体大腺瘤发生机制、生物学行为之间的联系。结果40例垂体大腺瘤中均发现PTTG的表达显著高于垂体微腺瘤组（P〈0．01）。在侵袭性垂体大腺瘤中，PTTG的表达显著高于非侵袭性垂体大腺瘤（P〈0．01）。PTTG的表达与大腺瘤向鞍上生长的高度和向海绵窦侵袭性生长的程度显著相关（P〈0.05）。结论PTTG表达增高与垂体大腺瘤的生长以及肿瘤的侵袭性密切相关。PTTG的表达水平及结合影像学资料可以为患者预后及术后的辅助治疗提供可靠的判断依据。     

ACTH 4-9 analogue (Org 2766) in depressed elderly patients. I. Effect on depressed mood

Sixty-two moderately depressed elderly in- and out-patients were administered 80 mg ACTH 4-9 analogue (Org 2766) or placebo daily for 4 weeks in a double-blind interindividual comparison. Patients were rated on days 0, 15 and 29 with the Hamilton Psychiatric Depression Rating Scale (HPDRS), The Hamilton Anxiety Scale (HAS) and the Sandoz Clinical Assessment Geriatric Scale (SCAG). After 4 weeks 23 patients crossed over to the opposite treatment for a further 2 weeks. A statistically significant reduction in the severity of depression and anxiety was found in both treatment groups. No statistically significant differences were found between the treatment groups, when comparing scores for each item and total scores of HPDRS, HAS and SCAG on day 0, 15 or 29. Nor were there any statistically significant differences after the cross-over. Somatic examination and laboratory screenings before and at the end of the study did not reveal any pathological changes. No side effects were recorded.     

Effects in vitro of New Growth Hormone Releasing Peptide (GHRP-1) on Growth Hormone Secretion from Ovine Pituitary Cells in Primary Culture

Continuous perifusion of pituitary cells was used to study the effects of a newly synthesized GHRP (GHRP-1 or KP 101) on growth hormone (GH) secretion from ovine pituitary cells and these have been compared to effects of growth hormone-releasing factor (GRF) and the original growth hormone-releasing peptide (GHRP-6). GH was continuously released at a constant rate during perifusion and secretion was increased by KP 101, GHRP-6 and GRF in a dose-dependent manner. The half-maximal effective dose of KP 101 and GHRP-6 was 10^?7 M, an order of magnitude higher than that for GRF. The maximal effects of KP 101 and GHRP-6 were similar but significantly less than the maximal effect of GRF. Blockade of calcium channels with Cd²⁺ (2 mM) totally and reversibly abolished the releasing effects of all three peptides. Like GHRP-6, the GH release induced by KP 101 was not affected by a GRF antagonist ([Ac-Tyr¹, D-Arg²]-GRF 1–29, 1 μM) which significantly reduced the effect of GRF on GH release. For each peptide, the response to a second application (1 h after the first application) was lower than the first response. When GRF (or KP 101, GHRP-6) was applied first and then KP 101 or GHRP-6 (or GRF) given 1 h later, the second response was not attenuated. Only a small additive effect on the release of GH by GRF was obtained by the co-administration of either KP 101 or GHRP-6. This result was achieved with maximal doses of the peptides, but not with half-maximal doses. These data indicate that KP 101 has similar potency and GH releasing properties to GHRP-6 but both are less potent than GRF. There is no synergistic effect of the peptides with GRF and only a small additive effect of KP 101 or GHRP-6 on GRF-stimulated GH release from ovine pituitary cells in vitro. KP 101 stimulates GH release by a mechanism that involves a common step employed also by GRF and GHRP-6, an increase in calcium influx. In addition, our data strongly suggest that KP 101 like GHRP-6 do not act through the GRF receptor and that there is no cross-desensitization the GRF elicited response.     

Targeted cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH) only transiently decreases the gene expression of pituitary receptors for LH-RH

A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.     

The effects of an ACTH (4–9) analogue on development of cisplatin neuropathy in testicular cancer: A randomized trial

The efficacy of the ACTH (4–9) analogue Org 2766 in the prevention of subclinical cisplatin neuropathy was assessed in a double-blind placebo-controlled multi-centre study in patients with testicular cancer or adenocarcinoma of unknown primary. Forty-two patients received at least four cycles of cisplatin (100 Mg/M² per cycle), together with subcutaneous injections of Org 2766 (2 mg/day for 5 consecutive days) or placebo. Vibratory threshold was used as a measure of neuropathy. For each individual patient, the influence of cisplatin on vibratory perception was quantified by the slope of the regression line between the natural logarithm of the vibratory thresholds and the number of cycles. From the slopes, the proportional increase of vibratory threshold per cycle of cisplatin was calculated. On average, vibratory tresholds increased by 42% with each cycle of 100 mg/m² of cisplatin in the placebo group, and by 19% during treatment with Org 2766. The influence of cisplatin on vibratory thresholds was highly significant in the placebo group (P < 0.0001), and of borderline significance in the group treated with Org 2766 (P = 0.06). The difference in slopes between the two groups was of borderline statistical significance (Wilcoxon's two-sample test:P = 0.06; analysis of variance:P = 0.04). These results show that Org 2766 cannot completely prevent cisplatin neuropathy in young men with testicular cancer, but nerve damage may be ameliorated by the use of this ACTH (4–9) analogue.     

Neurotrophic ACTH4–9 analogue therapy normalizes electroencephalographic alterations in chronic experimental allergic encephalomyelitis

Chronic experimental allergic encephalomyelitis (CEAE) is an established experimental model for multiple sclerosis (MS). The demyelinating lesions in the white matter of the central nervous system observed in CEAE and in MS are accompanied by various neurophysiological alterations. Among the best defined electrophysiological abnormalities are the changes in event-related potentials, in particular evoked potentials involving the spinal cord, i.e. motor and sensory evoked potentials. Less familiar are the changes observed in the electroencephalogram of CEAE-affected animals, which are also encountered in the human equivalent, MS. In the present experiment we evaluated the therapeutic value of a neurotrophic peptide treatment [H-Met(O₂)-Glu-His-Phe-d -Lys-Phe-OH, an ACTH_4–9 analogue] and its effect on the delayed flash visual evoked potentials (VEP) and power spectra of the electroencephalogram, during a 17-week follow-up of CEAE. CEAE animals treated with the neurotrophic peptide were protected against the development of neurological symptoms during the course of the demyelinating syndrome. VEPs of animals suffering from CEAE showed a delay of the latencies of the late components which was significantly counteracted by peptide treatment. The peak-to-peak amplitude of the VEP afterdischarge recorded from CEAE animals was significantly increased during the course of CEAE and correlated closely with the progression of the myelinopathy. Furthermore, CEAE animals showed an increase of electroencephalogram (EEG) beta activity of up to 500% as compared with the age-matched control group. This increase in beta power mainly consisted of a prevailing 20–21 Hz peak, a frequency that normally is not dominant in control EEG recordings of the rat during passive wakefulness. All these electrophysiological phenomena were absent in ACTH_4–9 analogue-treated animals. The present findings underscore the potential importance of a neurotrophic peptide treatment in the pharmacotherapy of central demyelinating syndromes, and possibly of MS.     

垂体瘤转化基因、FGF-2在垂体大腺瘤中的表达及其临床意义

目的:探讨垂体大腺瘤中PTTG和FGF 2的表达与肿瘤生物学行为的关系。方法:用免疫组化技术(LSAB法)分别检测垂体大腺瘤(4 0例)和垂体微腺瘤(11例)中PTTG和FGF 2的表达。结合临床影像分级资料,分析PTTG和FGF 2的表达与大腺瘤生物学行为的关系及临床意义。结果:40例垂体大腺瘤中均发现PTTG和FGF 2的表达增高,两者的表达水平均高于其在垂体微腺瘤中的表达(t=3 .845 ,P <0 .0 1;t =4.3 81,P <0 .0 1)。PTTG和FGF 2在垂体大腺瘤中的表达水平显著相关(r =0 .64 0 ,P <0 .0 1)。PTTG的表达与大腺瘤侵袭性生长的程度显著相关(P <0 .0 5 )。结论:PTTG上调FGF 2的表达机制在促进垂体大腺瘤的生长过程中发挥了重要的作用,并有可能为临床预后的评估提供可靠的依据。     

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca²⁺]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca²⁺]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 ± 10 to 214 ± 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 ± 5.4 to 73.2 ± 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6–27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca²⁺]i rise in cells obtained from 7 NFPA (from 110 ± 34 to 151 ± 40 nM [Ca²⁺]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 μM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca²⁺ as well as blockade of L-type Ca²⁺ channels by dihydropyridine antagonists abolished [Ca²⁺]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.     

ACTH 4-9 analogue (Org 2766) in depressed elderly patients. II. Effect on memory and vigilance

In a double-blind interindividual comparison 80 mg of an ACTH 4-9 analogue (Org 2766) or placebo was administered daily to 49 elderly depressed in- and out-patients for 4 weeks. 20 patients then changed to the opposite treatment in a cross-over study for a further 2 weeks. All patients were tested for memory on days 0 and 29 with a battery consisting of the 30 World-Pair Test, the 30 Figure-Test and the 30 Personal-Facta Test. Three scores were obtained from each test, immediate memory score (IMS), delayed memory score (DMS) and their difference, forgetting score (FS). Cross-over patients were tested for vigilance on days 0, 29 and 43 in an apparatus testing ability to rapidly detect and react to specific minor changes at random intervals. Org 2766 had no better effect than placebo on learning, consolidation, or retrieval of memorized material in elderly depressed patients. Statistically significant fewer target misses in the test of vigilance suggest a higher degree of sustained attention after treatment with Org 2766.     

Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTH-secreting pituitary adenomas in vitro

Ghrelin is a brain-gut peptide with wide-ranging endocrine, metabolic, cardiovascular and neural effects. Ghrelin, like its synthetic counterparts, the growth hormone (GH) secretagogues, has been shown to markedly stimulate adrenocorticotrophic hormone (ACTH) and cortisol secretion in humans and the ACTH-releasing effect of GH secretagogues is even greater in patients with pituitary ACTH-secreting tumours. Furthermore, these tumours synthesize ghrelin itself, suggesting an intrapituitary ghrelin circuit. The aim of the present study was to evaluate the effect of ghrelin on ACTH secretion by human pituitary corticotroph tumours in vitro to test the functionality of this circuit. Nine ACTH-secreting pituitary tumours (four microadenomas, five macroadenomas) were collected during surgery and incubated with 10-100 nM human ghrelin or with 10 nM human corticotrophin-releasing hormone (CRH). Control experiments were performed in rat anterior pituitary primary cultures. ACTH secretion was assessed after 4 h and 24 h incubation by immunometric assay. After 4 h of incubation with ghrelin, medium ACTH concentrations were two- to ten-fold higher compared to ACTH concentrations in unstimulated wells. The ACTH-releasing effect of ghrelin was significantly less than the response elicited by 10 nM CRH (up to 40-fold) Similar results were obtained after 24 h of incubation and a superimposable response pattern was observed in rat anterior pituitary primary cultures. The present study demonstrates that the endogenous GH secretagogue, ghrelin, stimulates ACTH secretion directly from human tumoural corticotrophs, as well as from normal rat pituitary, and indicates that the marked ACTH release elicited by ghrelin in patients with Cushing's disease in vivo is due, at least in part, to its action on the pituitary tumour. However, the reversal of the response pattern reported in vivo, with ghrelin proving a lesser stimulant than CRH in vitro, suggests that additional, suprapituitary mechanisms are involved in the in vivo response. Moreover, these data uphold the concept of a functional intratumoural ghrelin paracrine circuit in human corticotroph adenomas.     

Pituitary afferents originating in the paraventricular organ (PVO) of the goldfish hypothalamus

The diencephalon of nonmammalian vertebrates contains aminergic perikarya situated beneath the ependyma lining the third ventricle, known as the paraventricular organ (PVO). Catecholamines were visualized in the goldfish forebrain by formaldehyde-glutaraldehyde-induced fluorescence. Neuronal somata containing catecholamines were found in three paraventricular nuclei--the nucleus recessus posterioris (NRP), the nucleus recessus lateralis (NRL), and the nucleus posterioris paraventricularis (NPPv)--which may be considered to constitute the PVO of the goldfish. Lesion-degeneration investigations were conducted to determine whether the PVO contributes to the innervation of the goldfish pituitary. Following electrothermic lesions of the NRP, degenerating axons and nerve terminals were observed in the rostral pars distalis and in the proximal pars distalis, but not in the neuro-intermediate lobe of the pituitary. Following lesions of the NRL or of the NPPv, degenerating axons and nerve terminals were observed in the rostral pars distalis, the proximal pars distalis, and in the neurointermediate lobe. These observations demonstrate that the PVO is a source of pituitary afferents in the goldfish and suggest that the PVO is a source of the catecholaminergic innervation of the teleost pituitary.     

Anterior pituitary gland synthesises dopamine from l‐3,4‐dihydroxyphenylalanine (l‐dopa)

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l ‐3,4‐dihydroxyphenylalanine (l ‐dopa). Accordingly, we investigated the expression of aromatic l ‐amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l ‐dopa and its release in vitro. Then, we explored the effects of l ‐dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l ‐dopa. Also, the presence of l ‐dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l ‐dopa. In addition, l ‐dopa reduced corticotrophin‐releasing hormone‐stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD‐1015. Moreover, DA formation from l ‐dopa increased apoptosis and decreased proliferation. However, in the presence of NSD‐1015, l ‐dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l ‐dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l ‐dopa exerts direct actions independently from its metabolisation to DA.     

Studies of the Localisation of Kisspeptin Within the Pituitary of the Rhesus Monkey (Macaca mulatta) and the Effect of Kisspeptin on the Release of Non-Gonadotropic Pituitary Hormones

Kisspeptin neurones in the arcuate nucleus play a pivotal role in the regulation of hypothalamic gonadotrophin‐releasing hormone (GnRH) secretion in higher primates. To examine whether kisspeptin also influences the function of the primate pituitary directly, two experiments were performed in adult male rhesus monkeys. First, the distribution of kisspeptin‐containing cells in the pituitary was described using fluorescence immunohistochemistry. Second, the secretion of non‐gonadotrophin adenohypophysial hormones [growth hormone (GH), prolactin and thyroid‐stimulating hormone (TSH)] and cortisol in response to i.v. kisspeptin administration was examined. Eight animals were deeply anaesthetised and transcardially perfused with 4% paraformaldehyde. Fluorescence immunohistochemistry was performed on 25‐μm thick free‐floating pituitary sections to localise immunopositive kisspeptin cells and to examine their relationship with immunostaining for luteinising hormone (LH), follicle‐stimulating hormone, GH, prolactin, α‐melanocyte‐stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH) and GnRH. Kisspeptin cells were found in the intermediate lobe of all animals and, in four monkeys, this neuropeptide was also observed in cells scattered in the periphery of the anterior lobe. Kisspeptin colocalised with α‐MSH‐immunopositive cells in the intermediate lobe and, in 50% of the monkeys, with ACTH‐immuunopositive cells in the periphery of the adenohypophysis. There was no evidence for colocalisation of kisspeptin with gonadotrophs, somatotrophs or lactotrophs. Beaded kisspeptin axons were observed in the neural lobe. In addition, assay of plasma samples that had been collected for a previous study documenting kisspeptin‐10‐induced LH release in male monkeys revealed that kisspeptin administration failed to influence circulating concentrations of GH, prolactin, TSH and cortisol. Release of all four of these non‐gonadotrophic hormones, however, was stimulated markedly by NMDA, which is considered to act centrally. Although the morphological findings obtained in the present study are consistent with the notion that kisspeptin may act directly at the level of the pituitary, the nature of such an action remains to be defined.     

Inhibitory Effect of Prepro‐Thyrotrophin‐Releasing Hormone (178–199) on Adrenocorticotrophic Hormone Secretion by Human Corticotroph Tumours

Prepro‐thyrotrophin‐releasing hormone (TRH) (178–199), a 22‐amino acid cleavage product of the TRH prohormone, has been postulated to act as an adrenocorticotrophin hormone (ACTH)‐release inhibitor. Indeed, although in vitro evidence indicates that this peptide may inhibit basal and stimulated ACTH secretion in rodent anterior pituitary primary cultures and cell lines, not all studies concur and no study has as yet evaluated the effect of this peptide in Cushing’s disease. The present study aimed to test the effect of preproTRH(178–199) in human tumoural corticotrophs. Twenty‐four human ACTH‐secreting pituitary tumours (13 macroadenomas, 11 microadenomas) were collected during surgery and incubated with 10 or 100 nm preproTRH(178–199). ACTH secretion was assessed after 4 and 24 h of incubation by immunometric assay and expressed relative to levels observed in control, unchallenged wells (= 100%). Parallel experiments were performed in rat anterior pituitary primary cultures. A clear inhibition of ACTH secretion at 4 and 24 h was observed in 12 specimens (for 10 nm ppTRH: 70 ± 4% control at 4 h and 83 ± 5% control at 24 h; for 100 nm ppTRH: 70 ± 4% control at 4 h and 85 ± 5% control at 24 h), whereas a mild and short‐lasting stimulatory effect was observed in three tumours and no changes in ACTH secretion in the remaining nine tumoural specimens. The inhibitory effect of preproTRH(178–199) was more evident in macroadenomas and significantly correlated with sensitivity to dexamethasone inhibition. Significant inhibition of ACTH secretion by preproTRH(178–199) in rat pituitary cultures was observed after 24 h of incubation. The present study conducted in a large series of human corticotroph tumours shows that preproTRH(178–199) inhibits tumoural ACTH secretion in a sizable proportion of specimens, in close relation to the size of the tumour and its sensitivity to glucocorticoid negative feedback. This appears a promising avenue of research and further studies are warranted to explore the full scope of preproTRH(178–199) as a regulator of ACTH secretion.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Effects of Chronic Octreotide Treatment on GH Secretory Dynamics and Tumor Growth in Rats Bearing an Ectopic Somatotroph (GC) Tumor

The effects of octreotide, a long-acting somatostatin agonist selective of the sstr2/sstr3/sstr5 receptor subtypes, on ectopic GH secretion and tumor growth were investigated in Wistar-Furth female rats implanted with GH secreting (GC) cells which express mostly somatostatin receptors of the sstr1 and sstr2 subtypes. Octreotide dose dependently inhibited thymidine incorporation (?57%) and GH secretion (?41%) from GC cells in culture. In vivo, 6 weeks after GC cell implantation, plasma GH, IGF-1 and insulin levels were highly elevated. Cluster analysis of GH secretory dynamics revealed that GH secretion was less pulsatile in GC-implanted than in control animals. Furthermore, in GC-implanted animals, passive immunization either with SRIH or GHRH antisera did not affect GH plasma levels. Three weeks after GC cell implantation, when tumors became palpable, octreotide (1 μg/h/kg BW) or saline was infused constantly for three weeks by osmotic minipumps. In octreotide treated rats, GH, IGF-1 and insulin levels were not different from sham-implanted animals and tumors weight were reduced by 80%. High affinity somatostatin binding sites were found in equivalent amounts on tumors from octreotide-treated or saline-treated animals. These findings indicate that GH secretion in GC-rats is mainly derived from the tumors and independent of hypothalamic control and that octreotide reduces both GH secretion and tumor growth. We conclude that the GC-implanted rat represents a good animal model to test the antisecretory and antitrophic properties of somatostatin analogs in vivo.     

Pituitary Adenylate Cyclase Activating Peptide-38 (PACAP-38), PACAP-27, and PACAP Related Peptide (PRP) in the Rat Median Eminence and Pituitary

Pituitary adenylate cyclase activating peptide (PACAP) is a member of the vasoactive intestinal peptide-like peptide family. It is found in the hypothalamus, where the PACAP precursor is processed to form PACAP-38, the C-terminal truncated PACAP-27 and PACAP related peptide (PRP). Both PACAPs are potent stimulators of anterior pituitary adenylate cyclase activity, but the physiologically relevant anatomical sources of PACAP and possible importance of PRP in this regard are poorly understood. Using immunocytochemistry with epitope-specific antisera, we now show that PACAPS38, PACAP27- and PRP-positive nerve fibres are all present in the rat median eminence. The major immunoreactive species present was PACAP38. Numerous PACAP38-immmunoreactive nerve fibres were observed in the internal layer and a few were present in the posterior pituitary lobe. The external layer of the median eminence contained a few PACAP-38-immunoreactive fibres and PACAP-38-positive nerve terminals were rarely seen in the perivascular portal spaces. Surprisingly, delicate PACAP-38-positive nerve fibres were identified in the anterior pituitary lobe intermingled between the pituitary cells although none of the secretory pituitary cells contained immunoreactive PACAP38, PACAP27 or PRP and preproPACAP mRNA was not detected in the gland by Northern blotting or in situ hybridization. PACAP-27- and PRP-immunoreactive nerve fibres and terminals were found in the same locations as PACAP-38 although generally in lower numbers. Specific radioimmunoassays and HPLC revealed that PACAP-38 accounts for the vast majority of the adenohypophyseal PACAP-immunoreactivity, whereas PACAP-27 and PRP were found in low to undetectable concentrations. In primary cultures of rat pituitary cells and in the clonal gonadotrope-derived aT3-1 cell line, PACAP-27 and PACAP-38 were equipotent stimulators of cAMP accumulation, whereas PRP was ineffective. We conclude that the distribution of PACAP-imrnunoreactive nerve fibres in the hypothalamus of the adult male rat is not that expected for a classic releasing factor suggesting that other sources of PACAP are relevant for stimulation of anterior pituitary cells or that the hypothalamic PACAP system is activated under specific endocrine or developmental conditions.     

Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) on Prolactin, Luteinizing Hormone and Growth Hormone Secretion in the Ewe

This study was undertaken to investigate the roles of PACAP and VIP in the control of pituitary hormone secretion in the ewe. The first experiment was designed to identify any direct effects at the level of the pituitary and was conducted during the luteal phase of a prostaglandin-synchronized oestrous cycle. PACAP (0.008, 0.04, 0.2 and 1.0 nmol/min) or VIP (0.06, 0.2, 0.6 and 1.8 nmol/min) was infused into the carotid artery over a 10 min period. Blood samples were taken before and after the infusions so that plasma PRL, LH and GH concentrations could be measured. Blood pressure was also monitored to determine if the doses used were biologically active. In no case was an effect on hormone secretion observed. In contrast, the highest dose of each peptide induced an increase in heart rate to almost three-fold the resting value. Although both peptides are active in vivo, this result suggests that neither peptide has a direct effect on hormone release from the pituitary of prostaglandin-synchronized ewes. In a second experiment, we investigated whether the peptides had central effects on hormone secretion. Intracerebroventricular (ICV) injection of PACAP or VIP at the dose 10nmol was tested in ovariectomized ewes. After injection, PACAP suppressed PRL and GH secretion so that plasma hormone concentrations from 1–3 h after injection were significantly different from the control (P<0.05 for PRL, P<0.01 for GH). In addition, PACAP significantly reduced mean LH concentration (P<0.05) and LH pulse frequency (P<0.01). A similar suppressive effect on LH secretion was also observed after ICV injection of VIP (P<0.05 for both parameters), although PRL and GH release were not affected. These results suggest a possible role for PACAP in the neuroendocrine control of PRL, GH and LH secretion in sheep. In addition, VIP may be involved in the control of LH secretion. In contrast, there is no evidence to suggest that either peptide is a hypophysiotropic factor for PRL, LH or GH in prostaglandin-synchronized ewes.