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.

Accumulation of rat pineal serotonin N-acetyltransferase mRNA induced by pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide in vitro.

In mammals, pineal melatonin secretion is under the control of adrenergic and peptidergic inputs regulating serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase; AA-NAT) activity. In this study, the accumulation of AA-NAT mRNA induced by norepinephrine (NE) and peptides of the secretin superfamily (pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), growth hormone releasing factor (GRF), secretin) was investigated by a new quantitative reverse transcription-PCR (RT-PCR) assay. We demonstrated that PACAP was the most potent peptide to increase the expression of AA-NAT mRNA and to induce cAMP production in rat pinealocytes. VIP was also able to elevate the AA-NAT mRNA level and cAMP efflux in a dose-dependent manner; however, it was six- and threefold, respectively, less potent than PACAP. The maximal values of AA-NAT mRNA level after PACAP and VIP exposures were similar (523.1 +/- 52.5 amol to 640.7 +/- 68.8 amol vs 461.5 +/- 54.3 amol to 579.2 +/- 72.4 amol). These saturable peak values were approximately five- to eightfold less than that after NE (3.0 +/- 0.3 fmol to 3.6 +/- 0.4fmol). GRF and secretin were less potent than VIP in inducing AA-NAT gene expression and cAMP efflux. These data suggest that the peptides act mostly on VIP(1)/PACAP (VPAC(1)) receptors of pinealocytes with different affinity. The peak cAMP efflux always preceded the elevation of AA-NAT gene expression during the 3-h infusion of VIP or NE. The cAMP efflux had declined by the time of onset of maximal AA-NAT gene expression, but remained significantly higher than its basal values. Our data indicate that even a submaximal level of cAMP is sufficient for maintaining the maximal AA-NAT mRNA accumulation. These findings show that, in addition to NE, PACAP and VIP may have an important role in the regulation of AA-NAT mRNA levels in rat pinealocytes.

Regulation of prostate-specific antigen (PSA) gene expression and release in LNCaP prostate cancer by antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide.

BACKGROUND: Prostate-specific antigen (PSA) is the best tumor marker for diagnosis and prognosis of prostatic carcinoma. The secretion of PSA from LNCaP human prostate cancer cells is influenced by acute stimuli such as vasoactive intestinal peptide (VIP), growth hormone-releasing hormone (GHRH), and chronic stimuli like androgens. METHODS: To study the regulation of basal and VIP/GHRH or androgen-stimulated secretion from LNCaP cells, we used a superfusion system, which allowed us to simultaneously measure PSA gene expression, PSA secretion, and cAMP release from the same cancer cells. LNCaP cancer cells were also implanted orthotopically into nude mice. RESULTS: VIP (30 pM-3 nM), GHRH (3 nM-300 nM), and dihydrotestosterone (100 nM) induced a significant increase in PSA gene expression, PSA secretion, and cAMP release. The dose and time-dependent effects of peptides were manifested only in the presence of androgens. At the end of continuous stimulation of cells with 1 nM VIP for 2 hr, large amounts of stored immunoreactive PSA still remained in the cells. Adenylate cyclase activator, forskolin (FSK), significantly increased PSA secretion and gene expression, and potassium, which causes nonspecific depolarization of membranes, augmented gene expression, and secretion of PSA, but did not influence cAMP release. This suggests that PSA secretion is regulated by cAMP-dependent as well as cAMP-independent pathways. In superfusion system, stimulatory effects of VIP and GHRH on PSA secretion were inhibited by VIP antagonist JV-1-53, and less by GHRH antagonist JV-1-38. In cell cultures, JV-1-38 had a stronger inhibitory effect on proliferation, indicating an involvement of the recently discovered tumoral GHRH receptors in this process. In nude mice, with orthotopically implanted LNCaP cancer cells, GHRH antagonist JV-1-38 alone or androgen ablation by castration had no effect on tumor growth and PSA levels. However, castration combined with treatment with GHRH antagonist, significantly decreased tumor growth and PSA secretion. CONCLUSIONS: Our findings suggest that the secretion of PSA is regulated rather than constitutive, contrary to previous reports. In addition, the effect of GHRH and VIP antagonists on PSA secretion from prostate cancer cells is not correlated with their antiproliferative action.

Antiproliferative actions of growth hormone-releasing hormone antagonists on MiaPaCa-2 human pancreatic cancer cells involve cAMP independent pathways.

We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10-30 microM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [(125)I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH.

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 microg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 microg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1--0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

Peptide analogs in the therapy of prostate cancer.

The use of peptide analogs in the therapy of prostate cancer is reviewed. The preferred primary treatment of advanced androgen-dependent prostate cancer is presently based on the use of depot preparations of LH-RH agonists. This treatment is likewise recommended in patients with rising PSA levels after surgery or radiotherapy. LH-RH agonists with or without antiandrogens can be also utilized prior to or following various local treatments in patients with clinically localized prostate cancer and at high risk for disease recurrence. LH-RH antagonists like Cetrorelix are in clinical trials. However, most patients with advanced prostatic carcinoma treated by any modality of androgen deprivation eventually relapse. Treatment of relapsed androgen-independent prostate cancer remains a major challenge, but new therapeutic modalities are being developed based on antagonists of growth hormone-releasing hormone (GH-RH) and bombesin, which inhibit growth factors or their receptors. Another approach consists of cytotoxic analogs of LH-RH, bombesin, and somatostatin containing doxorubicin or 2-pyrrolinodoxorubicin, which can be targeted to receptors for these peptides found in prostate cancers and their metastases. These cytotoxic analogs inhibit growth of experimental androgen-dependent or -independent prostate cancers and reduce the incidence of metastases. A rational therapy with peptide analogs could be selected on the basis of receptors present in biopsy samples. The approaches based on peptide analogs should result in a more effective treatment for prostate cancer.

Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1-38 (20 microgram/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with (125)I-labeled GHRH antagonist JV-1-42, we demonstrated the presence of specific high-affinity (K(d) = 0.25 +/- 0.03 nM) binding sites for GHRH with a maximal binding capacity (B(max)) of 70.2 +/- 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of (125)I-JV-1-42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with (125)I-VIP (K(d) = 0.89 +/- 0.14 nM; B(max) = 183.5 +/- 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His(1),(125)I-Tyr(10), Nle(27)]hGHRH(1-32)NH(2). Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of (125)I-JV-1-42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers.

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV(1) and SV(2) are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV(1) is identical to that of human pituitary GHRH-R, whereas in SV(2) exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4-13 in SV(1) is identical to that of pituitary GHRH-R. SV(2) may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Potentiation of the inhibitory effect of growth hormone-releasing hormone antagonists on PC-3 human prostate cancer by bombesin antagonists indicative of interference with both IGF and EGF pathways.

BACKGROUND: In view of the involvement of various neuropeptides and growth factors in the progression of androgen-independent prostate cancer, we investigated the effects of antagonists of growth hormone-releasing hormone (GHRH) alone or in combination with an antagonist of bombesin/gastrin-releasing peptide (BN/GRP) on PC-3 human prostate cancers. METHODS: Nude mice implanted with PC-3 tumors received GHRH antagonists MZ-5-156 or JV-1-38, each at 20 microgram/day s.c. In experiment 2, treatment consisted of daily injections of JV-1-38 (20 microgram), BN/GRP antagonist RC-3940-II (10 microgram), or a combination of JV-1-38 and RC-3940-II. Serum IGF-I levels, expression of mRNA for IGF-II, and characteristics of BN/GRP and EGF receptors in tumor tissue were investigated. RESULTS: JV-1-38 induced a greater inhibition of tumor growth and suppression of IGF-II mRNA than MZ-5-156, both compounds causing a similar decrease in serum IGF-I. In experiment 2, JV-1-38 and RC-3940-II produced a comparable reduction in tumor volume (65% and 61%, respectively), but a combination of both antagonists augmented tumor inhibition to 75%. Combined treatment with JV-1-38 and RC-3940-II also led to a greater suppression of IGF-II mRNA (92%), as compared with JV-1-38 (72%) or RC-3940-II (77%). Serum IGF-I concentration was lowered only in mice treated with JV-1-38, while the downregulation of BN/GRP and EGF receptors was specific for groups receiving RC-3940-II. CONCLUSIONS: The inhibitory effects of GHRH antagonists on PC-3 human androgen-independent prostate cancer can be potentiated by concomitant use of BN/GRP antagonists. The combination of both types of analogs apparently interferes with both IGF and bombesin/EGF pathways, and might be clinically useful for the management of androgen-independent prostate cancer.

Antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide inhibit tumor proliferation by different mechanisms: evidence from in vitro studies on human prostatic and pancreatic cancers.

Antagonists of GH-releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) inhibit the proliferation of various tumors in vitro and in vivo, but a comparison of their antitumor effects and mechanisms of action has not been reported to date. We recently synthesized and characterized a series of analogs, some of which are primarily GHRH antagonists (JV-1-36, JV-1-38, and JV-1-42), whereas others are more selective for VIP receptors (VPAC-R; JV-1-50, JV-1-51, JV-1-52, and JV-1-53). LNCaP human prostatic cancer cells express VPAC-R, with predominant subtype 1 determined by RT-PCR. Our studies show that GHRH antagonists significantly inhibit the proliferation of both VPAC-R positive LNCaP cells (P < 0.001) and VPAC-R negative MiaPaCa-2 human pancreatic cancer cells cultured in vitro (P < 0.05 to P < 0.001). Growth inhibition of LNCaP cells is accompanied by a proportional reduction in prostate-specific antigen (PSA) secretion (P < 0.001). In a superfusion system, the inhibitory activities of the analogs on the rate of VIP and GHRH-induced PSA secretion correlate well with their VPAC-R binding affinities to LNCaP cell membranes. Antagonists more selective for VPAC-R display a stronger inhibition of inducible PSA release than GHRH antagonists, but have smaller effects or no effects on proliferation and PSA secretion in culture. Collectively, our findings demonstrate that the antiproliferative activity of the analogs on cancer cells is not correlated to their VPAC-R antagonistic potencies. Because GHRH antagonists inhibit the proliferation of LNCaP cells more powerfully than VPAC-R antagonists and also suppress the growth ofVPAC-R-negative MiaPaCa-2 cells, it can be concluded that their antiproliferative effect is exerted through a mechanism independent of VPAC-R.

Antagonists of growth hormone-releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice.

Since antagonists of growth hormone-releasing hormone (GH-RH) inhibit proliferation of various tumors, in this study we investigated the effects of GH-RH antagonists MZ-5-156 or JV-1-36 on growth of estrogen-independent MDA-MB-468 human breast cancers xenografted into nude mice. Both GH-RH antagonists administered at a dose of 20 microg/day induced regression of some and growth-arrest of other tumors, while control tumors continued to grow. After 5 weeks of therapy with MZ-5-156 or JV-1-36, final volume and weight of MDA-MB-468 tumors were significantly decreased (all p values < 0.001) and serum IGF-I levels as well as tumor IGF-I mRNA expression were reduced as compared with controls. High affinity binding sites for IGF-I were detected by the ligand binding method. Gene expression of human IGF-I receptors, as measured by the RT-PCR, was not significantly different in control and treated MDA-MB-468 tumors. In cell culture, IGF-I did not stimulate, GH-RH slightly stimulated, while MZ-5-156 and JV-1-36 inhibited proliferation of MDA-MB-468 cells known to possess defective insulin and IGF-I receptor signaling. The expression of mRNA for human GH-RH was found in five of 8 tumors treated with GH-RH antagonists, and in one of the five control tumors. These results suggest that GH-RH antagonists inhibit MDA-MB-468 breast cancers possibly through mechanisms involving interference with locally produced GH-RH.

Antagonistic actions of analogs related to growth hormone-releasing hormone (GHRH) on receptors for GHRH and vasoactive intestinal peptide on rat pituitary and pineal cells in vitro.

Peptide analogs of growth hormone-releasing hormone (GHRH) can potentially interact with vasoactive intestinal peptide (VIP) receptors (VPAC(1)-R and VPAC(2)-R) because of the structural similarities of these two hormones and their receptors. We synthesized four new analogs related to GHRH (JV-1-50, JV-1-51, JV-1-52, and JV-1-53) with decreased GHRH antagonistic activity and increased VIP antagonistic potency. To characterize various peptide analogs for their antagonistic activity on receptors for GHRH and VIP, we developed assay systems based on superfusion of rat pituitary and pineal cells. Receptor-binding affinities of peptides to the membranes of these cells were also evaluated by radioligand competition assays. Previously reported GHRH antagonists JV-1-36, JV-1-38, and JV-1-42 proved to be selective for GHRH receptors, because they did not influence VIP-stimulated VPAC(2) receptor-dependent prolactin release from pituitary cells or VPAC(1) receptor-dependent cAMP efflux from pinealocytes but strongly inhibited GHRH-stimulated growth hormone (GH) release. Analogs JV-1-50, JV-1-51, and JV-1-52 showed various degrees of VPAC(1)-R and VPAC(2)-R antagonistic potency, although also preserving a substantial GHRH antagonistic effect. Analog JV-1-53 proved to be a highly potent VPAC(1) and VPAC(2) receptor antagonist, devoid of inhibitory effects on GHRH-evoked GH release. The antagonistic activity of these peptide analogs on processes mediated by receptors for GHRH and VIP was consistent with the binding affinity. The analogs with antagonistic effects on different types of receptors expressed on tumor cells could be utilized for the development of new approaches to treatment of various human cancers.

Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities.

Some antagonists of human growth hormone-releasing hormone (hGH-RH) synthesized previously were shown to inhibit in vivo proliferation of various human cancers in nude mice. However, the activity of these analogs requires an increase to assure clinical efficacy. In an attempt to prepare hGH-RH antagonists with a high and protracted activity, we synthesized and biologically tested 22 antagonistic analogs of hGH-RH(1-29)NH2. The ability of the antagonists to inhibit hGH-RH-induced GH release was evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinity of the peptides to GH-RH receptors also was determined. All antagonistic analogs had the common core sequence [PhAc-Tyr1,D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), Nle27]hGH-RH(1-29)NH2 and contained Arg, D-Arg, homoarginine (Har), norleucine (Nle), and other substitutions. The following analogs were determined to have a high and/or protracted antagonistic activity: [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27, D-Arg29]hGH-RH(1-29)NH2 (JV-1-10), [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6, Abu15,Nle27,D-Arg28,Har29]hGH-RH(1-29)NH2 (MZ-6-55), [PhAc-Tyr1, D-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27,D-Arg28,Har29 ]hGH-RH(1-29)NH2 (JV-1-36), and [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Har9,Tyr(Me)10,Abu15, Nle27,D-Arg28,Har29]hGH-RH(1-29)NH2 (JV-1-38). Among the peptides tested, analog JV-1-36 showed the highest GH-RH antagonistic activity in vitro and also induced a strong and prolonged inhibition of GH release in vivo for at least 30 min. The antagonist JV-1-38 was slightly less potent than JV-1-36 both in vitro and in vivo but proved to be very long-acting in vivo, suppressing the GH-RH-induced GH release even after 60 min. High and protracted in vivo activities of these antagonists indicate an improvement over earlier GH-RH analogs. Some of these hGH-RH antagonists could find clinical applications in the treatment of cancers dependent on insulin-like growth factors I and II.

Adrenergic and peptidergic control of the regulation of cAMP efflux and melatonin secretion from perifused rat pineal gland.

Mammalian pineal gland receives peptidergic (e.g., vasoactive intestinal peptide [VIP]; peptide histidine isoleucine [PHI]; neuropeptide Y, NPY; substance P, calcitonin gene-related peptide [CGRP], arginine vasopressin [AVP] and oxytocin [OXT]) fibers in addition to sympathetic innervation. The dynamics of cAMP efflux and melatonin (MT) secretion were compared during the infusion of these peptides in our long-term perifusion system. VIP and PHI enhanced both pineal cAMP efflux and MT secretion in a dose-dependent manner (10 nM to 10 microM). However, the potency of PHI was slightly less. The peak of cAMP release always precedes that of MT production. The possible interactions between adrenergic and peptidergic compounds in the regulation of pineal cAMP efflux and MT secretion were also studied. VIP acts on specific peptidergic receptors, since its stimulatory effect could only be reduced by a VIP receptor antagonist. VIP has an additive effect at a lower (100 nM) concentration combined with norepinephrine (NE). NPY (100 nM) can completely block NE-induced MT secretion, but the decrease in cAMP efflux is less. However, NPY does not significantly influence VIP-stimulated cAMP efflux or MT secretion. These data suggest that NE, VIP, and NPY are differently involved in the cAMP and calcium signaling. The other neuropeptides are ineffective.

Melatonin secretion of the rat pineal gland in response to norepinephrine in different types of the anovulatory syndrome.

Earlier experimental results show the role of altered pineal function in the development of the constant estrous-anovulatory (CEA) state induced by neonatal androgen sterilization (NA) or in the spontaneously developed anovulatory syndrome in the aging rat (AG-CEA state). Since norepinephrinergic (NE) innervation of pineal gland represents the main stimulus for melatonin secretion in mammals, in the present experimental series, the reactivity of pineal tissue to NE was investigated in in vitro perifusion system in rats suffering from NA-CEA or AG-CEA state, using the model of pineal body deprived from neural inputs. The data indicate that the 'melatonin deficiency' observed in the 2 types of anovulatory syndrome (NA-CEA and AG-CEA states) is due to disturbance of norepinephrinergic innervation of the pineal gland rather than to deficient secretory capacity of pineal tissue.

Regulation of pineal melatonin secretion: comparison between mammals and birds.

The melatonin secretory pattern of the perifused rat and chicken pineal was compared in response to different lighting conditions and norepinephrine administration. The following main differences were observed. 1. Explanted (perifused) rat pineal showed, after a rapid initial surge, a steady continuous basal secretion of melatonin. This was independent from the day-night (light-dark) periods. Chicken pineal, however, showed a characteristic daily rhythm of melatonin production with peak in the dark and nadir in the light phase. 2. This daily rhythm could not be extinguished by keeping the pineal donor birds in constant light- or constant dark environment for at least 2 weeks immediately before sacrifice. 3. Short light impulse (5 min), applied in the middle of the dark phase, was ineffective in birds. Keeping the perifusion chambers in continuous light or darkness, however, depressed the amplitude of the circadian rhythm even in the next cycle. 4. Rat pineal responds to norepinephrine stimulation with a dose-related increase of melatonin release, independently from the phase of the day, while in the chicken, norepinephrine slightly inhibits both the diurnal and the nocturnal level of melatonin secretion. 5. It can be inferred that melatonin secretion of the mammalian pineal gland is primarily regulated by a peripheral (sympathetic) innervation. This is modulated, under in vivo circumstances, by different environmental factors, mainly by light conditions transmitted by neural mechanisms. In contrast, the primary secretory process of the avian pineal is based on an intrinsic circadian rhythm. This might be genetically coded or maintained by yet unknown neurohormonal mechanisms and/or external factors (e.g. magnetic fields). This fairly stable circadian rhythm is only modulated by environmental lighting conditions.

Synthesis and biological activities of highly potent antagonists of growth hormone-releasing hormone.

In the search for antagonists of human growth hormone-releasing hormone (hGHRH) with high activity, 22 analogs were synthesized by solid-phase methods, purified, and tested biologically. Within the N-terminal sequence of 28 or 29 amino acids of hGHRH, all the analogs contained D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), and Nle27 and most of them had Agm29 (agmatine) substituents. All the peptides, except one, were acylated at the N terminus with different hydrophobic acids--e.g., isobutyric acid (Ibu) or 1-naphthylacetic acid (Nac) in order to study the effect of N-terminal acylation on the antagonistic activity. In the superfused rat pituitary cell system, all the analogs inhibited more powerfully the GHRH-induced growth hormone (GH) release than the standard GHRH antagonist [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2. Antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-243), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGH RH-(1-29)NH2 (MZ-4-181), and [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27,Asp28]hGH RH-(1-28)Agm (MZ-4-209) inhibited GH release at 3 x 10(-9) M. Among these peptides, MZ-4-243, MZ-4-169, and MZ-4-181 were also long acting in vitro. Antagonist MZ-4-243 inhibited GH release 100 times more powerfully than the standard antagonist and was the most potent in vitro among GHRH antagonists synthesized. Analogs with high inhibitory effects in vitro were also found to have high affinities to rat pituitary GHRH receptors. In experiments in vivo, antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGHRH-(1-28 )Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), and [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHR H-(1-29)NH2 (MZ-4-181) induced a significantly greater inhibition of GH release than the standard antagonist. In view of their high antagonistic activity and prolonged duration of action, some of these antagonists of GHRH may find clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors.

Differences in hormone release patterns from the anterior pituitary and the pineal gland.

Patterns of hormone release from peptide- and monoamine-hormone secreting cells were compared. LH, GH and PRL secretion from rat anterior pituitary cells and melatonin secretion from the rat pineal gland fragments were studied in a dynamic, in vitro system. The following fundamental differences were found: 1. Adenohypophysial cells respond to a specific stimulus (releasing hormones) with hormone secretion within seconds. There is a 90-minute lag, however, between the beginning of specific stimulation (norepinephrine, 1 microM for 30 min) and the onset of melatonin (MT) release from the pineal cells. 2. Hormone secretion from the pituitary cells returns to the basal value five to 10 minutes after the stimulus has been stopped. Once initiated, MT release from the pineal lasts for five to seven hours even if the NE stimulus was stopped one hour before the beginning of the MT response. 3. A transitory increase in potassium concentration (by 50-100 mM) induces a sharp, distinct rise of hormone secretion from pituitary cells, similar in shape and size experienced as response to releasing hormone stimulation. Pineal cells do not respond to elevation of potassium concentration at all. 4. High concentration of tropic hormones can be extracted from pituitary cells at the end of the superfusion experiment. Their hormone content is equivalent to the amount released from non-stimulated cells in a 50 to 80 hours period (LH and GH cells). No significant quantity of MT can be extracted from non-stimulated, disintegrated pineal cells. This diversity in the control of hormone secretion from anterior pituitary and the pineal gland can be considered as a model for peptide and monoamine-hormone producing glands.

LH-RH analogue carrying a cytotoxic radical is internalized by rat pituitary cells in vitro.

The binding and internalization of a cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH), T-98 (agonist [D-Lys6]LH-RH linked to glutaryl-2-(hydroxymethyl)anthraquinone), by rat anterior pituitary cells was investigated. Analogue T-98 was bound to pituitary membrane binding sites for LH-RH with a high affinity (Kd = 1.2 nM) and was 17 times more potent in releasing luteinizing hormone (LH) from superfused rat pituitary cells than LH-RH. The labeling of this cytotoxic LH-RH analogue was carried out both with radioactive (125I) and nonradioactive iodine. Monoiodination of the Tyr5 residue of T-98 did not significantly affect its binding affinity but greatly decreased its LH-releasing activity to about 3% of the original value. Di-iodination in the same position lowered binding affinity twenty-threefold and further diminished LH-releasing potency. [125I]T-98 was found to bind very strongly to polystyrene, which precluded the use of regular tissue culture plasticware in our experiments. In pituitary cells cultured in glass vials, binding and internalization of [125I]T-98 were observed, which were time and temperature dependent, and which could be inhibited by excess unlabeled analogue. No enzymatic degradation of labeled T-98 was detected in the culture medium during the incubation. Our results indicate that T-98 is internalized by pituitary gonadotropes through receptor-mediated endocytosis. Because this new class of compounds was designed as anticancer drugs, our findings also suggest that this cytotoxic LH-RH agonist may also be internalized by LH-RH receptors present in breast, prostate, ovarian, and other tumors.

Effect of luteinizing hormone-releasing hormone analogs containing cytotoxic radicals on the function of rat pituitary cells: tests in a long term superfusion system.

A novel class of targeted chemotherapeutic agents based on cytotoxic compounds linked to LHRH analogs was tested in a long term superfusion system in order to determine their effects on different types of rat pituitary cells. The compounds investigated included two cytotoxic LHRH agonists, T-98 ([D-Lys6]LHRH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)) and T-107 ([D-Lys6]LHRH linked to doxorubicin (DOX) through glutaric acid spacer), and two cytotoxic LHRH antagonists T-121 and T-144, both containing two residues of G-HMAQ. The analogs were infused for 24 h at pharmacological (micromolar) concentrations. The secretions of LH, GH, and PRL cells after a short (2-h) and a long (20-h) recovery period following the treatment were compared to those before the analog infusion. All four cytotoxic LHRH analogs selectively decreased the stimulated LH response, while basal secretions of LH, GH, and PRL as well as the stimulated release of GH and PRL were not influenced in spite of the high doses applied and the long duration of exposure. The inhibitory effects of cytotoxic agonists and antagonists were significantly greater than those of their parent peptides. Equimolar concentrations of cytotoxic compounds alone (DOX or G-HMAQ), however, caused functional damage to all types of cells tested. To evaluate the mechanisms of the observed inhibitory actions, we compared the amount of LH released in response to 1) a specific stimulus to receptors (3 nM LHRH), 2) membrane depolarization with 100 MM KCl, not involving the receptor, and 3) the extraction of cells with 0.01 N HCl at the end of experiment. Cytotoxic LHRH agonists and their carriers caused depletion of LH pools, while cytotoxic LHRH antagonists or their parent peptides decreased the available binding sites for LHRH. The inhibitory effect of DOX on stimulated secretion of LH, GH, and PRL in our system could be due to an action on cell membranes. Our work indicates that in the pituitary cell superfusion system, targeted cytotoxic conjugates, based on LHRH analogs, selectively affect LH cells, in contrast to unconjugated cytotoxic compounds, which also damage GH and PRL cells.