Bombesin conjugated solid lipid nanoparticles for improved delivery of epigallocatechin gallate for breast cancer treatment.

Epigallocatechin-gallate (EGCG) is a potent anti-cancer therapeutic which effectively controls the growth of cancerous cells through a variety of different pathways. However, its molecular structure is susceptible to modifications due to cellular enzymes affecting its stability, bioavailability and hence, overall efficiency. In this study, we have initially encapsulated EGCG in the matrix of solid lipid nanoparticles to provide a stable drug carrier. To confer additional specificity towards gastrin releasing peptide receptors (GRPR) overexpressed in breast cancer, EGCG loaded nanoparticles were conjugated with a GRPR-specific peptide. In-vitro cytotoxicity studies showed that the peptide-conjugated formulations possessed greater cytotoxicity to cancer cell lines compared to the non-conjugated formulations. Further, in-vivo studies performed on C57/BL6 mice showed greater survivability and reduction in tumour volume in mice treated with peptide-conjugated formulation as compared to the mice treated with non-conjugated formulation or with plain EGCG. These results warrant the potential of the system designed in this study as a novel and effective drug delivery system in breast cancer therapy.

Peripheral injection of bombesin induces c-Fos in NUCB2/nesfatin-1 neurons.

As anorexigenic hormones bombesin and nucleobindin2 (NUCB2)/nesfatin-1 decrease food intake in rodents. Both hormones have been described in brain nuclei that play a role in the modulation of hunger and satiety, like the paraventricular nucleus of the hypothalamus (PVN) and the nucleus of the solitary tract (NTS). However, the direct interaction of the two hormones is unknown so far. The aim of study was to elucidate whether bombesin directly interacts with NUCB2/nesfatin-1 neurons in the PVN and NTS. Therefore, we injected bombesin intraperitoneally (ip) at two doses (26 and 32nmol/kg body weight) and assessed c-Fos activation in the PVN, arcuate nucleus (ARC) and NTS compared to vehicle treated rats (0.15M NaCl). We also performed co-localization studies with oxytocin or tyrosine hydroxylase. Bombesin at both doses increased the number of c-Fos positive neurons in the PVN (p<0.05) and NTS (p<0.05) compared to vehicle, while in the ARC no modulation was observed (p>0.05). In the PVN and NTS the number of c-Fos positive neurons colocalized with NUCB2/nesfatin-1 increased after bombesin injection compared to vehicle treatment (p<0.05). Moreover, an increase of activated NUCB2/nesfatin-1 immunoreactive neurons that co-expressed oxytocin in the PVN (p<0.05) or tyrosine hydroxylase in the NTS (p<0.05) was observed compared to vehicle. Our results show that peripherally injected bombesin activates NUCB2/nesfatin-1 neurons in the PVN and NTS giving rise to a possible interaction between bombesin and NUCB2/nesfatin-1 in the modulation of food intake.

The impact of diet and micronutrient supplements on the expression of neuroendocrine markers in murine Lady transgenic prostate.

BACKGROUND: Neuroendocrine (NE) differentiation (NED) in prostate cancer (PCa) is associated with morbidity and death; however, the underlying cause(s) promoting NED in PCa have yet to be determined. In this study, we examined the effect of both diet and micronutrient supplementation on the expression of NE markers using the Lady (12T-10) transgenic model of PCa. Lady (12T-10) transgenic animals develop advanced adenocarcinoma with NE characteristics that exhibits metastases in approximately 80% of cases. In this model a high fat diet has been shown to increase the severity of disease, while the use of micronutrients can inhibit this progression. METHODS: In this study we used immunohistochemical analysis to determine expression of the NE markers: chromogranin A (CgA), neuron-specific enolase (NSE), bombesin, parathyroid hormone-related peptide (PTHrP), neurotensin and serotonin in prostates of PCa-bearing Lady (12T-10) mice. RESULTS: High fat diet was correlated with significantly elevated expression of CgA and serotonin in prostate tissue of Lady (12T-10) mice. Addition of micronutrients to the control and high fat diet reproducibly elevated PTHrP and bombesin expression and suppressed NSE expression, while prostate tissue from the control diet supplemented with micronutrients exhibited significantly lower numbers of calcitonin- and neurotensin-positive cells. CONCLUSIONS: These results highlight the importance of dietary control in management of disease and identify differential changes in NE marker expression, which may be diagnostically viable in monitoring the impact of therapies on disease status.

Are neuropeptides important in arthritis? Studies on the importance of bombesin/GRP and substance P in a murine arthritis model.

Interference with the effects of neuropeptides may be of potential therapeutic value for the treatment of rheumatoid arthritis (RA). Two neuropeptides that can be discussed in this context are bombesin/gastrin-releasing peptide (BN/GRP) and substance P (SP). In order to obtain new information on the possible importance of these two peptides, the patterns of immunohistochemical expression of BN/GRP and SP and their related receptors in the mouse knee joint from healthy and arthritic mice were examined. Positive staining for GRP receptor and the SP preferred receptor (the neurokinin-1 receptor [NK-1 R]) was observed in articular chondrocytes. On the whole, there was a decrease in immunoreactions for both the GRP- and the NK-1 receptors in the articular chondrocytes in joints exhibiting severe arthritis. Staining for BN/GRP and GRP receptor was seen in the inflammatory infiltrates of the arthritic joints. New evidence for the occurrence of marked effects of BN/GRP concerning both the articular chondrocytes and the inflammatory process is obtained in this study. With these findings and previous observations of neuropeptide expression patterns and functions we discuss the possibility that interventions with the effects of BN/GRP, SP, and other neuropeptides might be worthwhile in RA.

Distributions of two chicken bombesin receptors, bombesin receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R) mRNAS in the chicken telencephalon.

Bombesin (BN)-like peptide receptors are known to be essential to the regulation of not only homeostasis, including feeding behavior, but also of emotional systems in mammal. Recently, two novel BN receptors, chicken BN-like peptide receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R), have been identified. Here, we report the localizations of these receptors' mRNAs in the chick brain through development using in situ hybridization. First, chBRS-3.5 mRNA signals were found in the dorsal ventricular ridge at embryonic day (ED) 9. Strong signals were observed in the hyperpallium accessorium, nidopallium and nucleus basorostralis pallii, and moderate signals were found in the hippocampus, cortex piriformis, hyperpallium intercalatum, area temporo-parieto-occipitalis, nucleus striae terminalis lateralis, nucleus olfactorius anterior and organum septi lateralis at ED16. This wide expression in the pallium persisted during posthatch periods. Abundant expressions in the hyperpallium, nidopallium, considered to be similar to the mammalian cortex, as well as in the hippocampus, indicate participation of these molecules in the processing of sensory information, motor function, learning and memory. Telencephalic areas devoid of chBRS-3.5 signals were the entopallium, arcopallium anterius, globus pallidus, nucleus intrapeduncularis, tuberculum olfactorius, nucleus septalis lateralis, hypothalamic and thalamic areas. In contrast to chBRS-3.5, chGRP-R mRNA signals were found in the pallidum at ED5 and 9. At ED16, chGRP-R mRNA signals were localized in the medial striatum and hypothalamus. GRP-R expression in the hypothalamic region was phylogenically conserved. Thus, chBRS-3.5 mRNA signals were distributed in a broader region and were more intense than chGRP-R mRNA. Taken together, chGRP-R and chBRS-3.5 mRNA occurred in similar regions of mammals that express GRP-R. BN/GRP-immunoreactive neurons and varicosities were found mainly in the pallium, especially in the hyperpallium accessorium and nidopallium, and this distribution coincided with that of chBRS-3.5 mRNA. This result suggests that the endogenous ligands for chBRS-3.5 were likely BN-like peptides produced in the pallium.

Bombesin-like peptides: candidates as diagnostic and therapeutic tools.

Bombesin (BBS) is proved to have a wide variety of the pharmacologic effects, including effects on the release of gastrointestinal hormones and control of gastrointestinal motility. More recently, the role of BBS in tumor growth, cellular proliferation and inflammation has attracted attention. There is evidence that increased BBS receptor expression may be considered as a specific marker for small-cell lung cancer, colorectal adenocarcinoma, gastric and pancreatic cancer, prostate, ovarian and breast cancer, neuroblastoma, renal cell carcinoma, malignant melanoma and thyroid carcinoma. BBS expression was found to be correlated with the histological grade of the tumor. Similarly, BBS treatment significantly improves the healing of chronic gastric ulcers and ameliorates the severity of burn- or colitis-induced gut injury. Although there is much complexity still to be elucidated to understand fully the physiologic and pathologic roles of BBS-like peptides several clinical or experimental trials have addressed that circulating or tissue levels of BBS-like peptides or their receptor expression may be used as diagnostic or prognostic markers of neoplastic disease, and incorporation of BBS receptor antagonists in the treatment of human cancer could provide substantial benefit to the cancer patients. Moreover, trophic, anti-ulcerogenic and anti-inflammatory actions of exogenous BBS make this peptide a potential supplement in minimizing or reversing tissue damage against several injurious challenges. In conclusion, based on the evidence summarized herein, related to the mitogenic and anti-inflammatory effects of BBS-like peptides, further investigations are needed to derive the benefit of BBS-like peptides in pharmacologic strategies.

A human gastric cancer cell line possesses a functional receptor for gastrin-releasing peptide.

Bombesin (BBS) exhibits diverse biological functions including those of neurotransmitter, regulator of gastrointestinal hormone release, and mitogen. Gastrin-releasing peptide (GRP, the mammalian equivalent of BBS) is found in mucosal cells of the gastric fundus and antrum. We determined whether a human gastric cancer cell line (SIIA) expresses a functional GRP-receptor (GRP-R). BBS increased intracellular calcium ([Ca2+]i), and a specific GRP-R antagonist, ([D-Phe6, Des-Met14]-BBS (6-14)-ethylamide), blocked BBS-induced increase in [Ca2+]i. SIIA cells possess GRP-R mRNA by reverse transcriptase-PCR. Furthermore, these cells possess an 80-kDa cell surface protein that specifically binds BBS with two high-binding affinities (Kd1 = 0.6 nM, Kd2 = 6.7 nM). These findings indicate that SIIA cells possess a GRP-R that is capable of physiological signal transduction, though the cellular response remains unknown.

Regulation of ingestion by CRF and bombesin-like peptides: distinct meal-related peptide level changes.

Bombesin (BN) and corticotropin-releasing factor (CRF) have both been shown to induce satiety in rats when injected centrally. The present study assessed temporal changes in the utilization of BN- and CRF-like peptides in relationship to feeding status, fluctuations that may indicate the physiological participation of these peptides in the regulation of feeding. Alterations in the endogenous levels of CRF- and BN-like peptides associated with the initial spontaneous meal of the nocturnal cycle were determined in 15 hypothalamic and extrahypothalamic brain nuclei in the following three groups of rats: 1) a preprandial group consisting of rats killed before feeding, 2) a prandial group consisting of rats killed during the meal, and 3) a postprandial group consisting of rats killed 8-12 min after the meal. Findings revealed site-specific changes in BN and CRF content during the course of a meal. During ingestion, levels of BN were significantly elevated at the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus and reduced at the nucleus accumbens. In the case of CRF, feeding-related alterations were observed at the lateral (LH) and ventromedial (VMH) hypothalamic nuclei and at the central nucleus of the amygdala (Ce). At the LH, CRF content decreased after feeding compared with preprandial levels. At the VMH, CRF levels were significantly elevated both before and after food intake compared with prandial levels. In contrast, at the Ce marked increases in CRF concentrations were observed during ingestion. These data demonstrate, for the first time, site-specific fluctuations of BN and CRF in relationship to the animal's feeding status and suggest that these peptides may play a role in the regulation of food intake.

Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes.

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)

NGF suppression of weight gain in adult female rats correlates with decreased hypothalamic cholecystokinin levels.

Effects of chronic intraventricular administration of nerve growth factor (NGF, 1 microgram qod for 21 days) on weight gain, hypothalamic neuropeptide levels and choline acetyltransferase (ChAT) activity were determined in adult female Wistar rats. Rats chronically treated with cytochrome c (cc) gained 163 g over the 21 day treatment schedule, whereas NGF-treated rats only gained 110 g. Thus, NGF-treated rats gained 53 g less; this change in weight gain is equivalent to approximately a 20% decrease of total weight gain compared to the cc-treated control rats. Chronic NGF treatment significantly decreased hypothalamic cholecystokinin (CCK) levels by 24% (P = 0.0070), but did not alter either hypothalamic neuropeptide Y (NPY) or bombesin (BOMB) levels (98% and 105% of cc-treated control levels, respectively). In addition, chronic NGF treatment did not significantly alter hypothalamic ChAT activity (95% of cc-treated control rats). The results of the present study suggest that NGF-induced decreases in weight gain are not the result of alterations of hypothalamic cholinergic function. However, it is possible that NGF-induced alterations of hypothalamic CCK synthesis and release may be involved in the NGF-induced decrease in weight gain.

Autoradiographic demonstration of 125I-Tyr4-bombesin binding sites on rat anterior pituitary cells.

Because several direct effects of bombesin related peptides on pituitary hormone release have been demonstrated, we chose to study the presence of bombesin binding sites in the adult male rat anterior pituitary in aggregate cell culture by autoradiographic localization of 125I-tyrosine4-bombesin (125I-Tyr4-BBN) binding and immunocytochemical localization of the anterior pituitary hormones. When aggregates were cultured in medium without hormonal supplements, the number of cells with detectable 125I-Tyr4-BBN binding was below 1%. In cell aggregates cultured in the presence of 1 nM estradiol (E2) 125I-Tyr4-BBN binding was detected on 5.4 +/- 0.8% of the cells after redispersion and on 5.8 +/- 1.1% of the cells in sections of paraffin embedded aggregates. The binding cell types were mainly lactotrophs and somatotrophs. The binding of 125I-Tyr4-BBN (3 or 5 nM) was specific because it was inhibited by the addition of an excess of unlabelled Tyr4-BBN or the bombesin receptor antagonist L686,095. In aggregates cultured in the presence of 1 nM E2 and 4 nM dex, the percentage of cells with detectable 125I-Tyr4-BBN binding was significantly lower than that in aggregates cultured in the presence of 1 nM E2 alone. Binding on somatotrophes almost completely disappeared. The present data show that specific 125I-Tyr4-BBN binding sites are present on anterior pituitary cells, are detectable mainly on a small subpopulation of lactotrophs and somatotrophs, and are affected by hormonal conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid alterations of hypothalamic and hippocampal bombesin-like peptide levels with feeding status.

It has been suggested that bombesin (BN)-like peptides may play a physiological role in the control of food intake. We studied the time course of changes in the levels of central BN-like peptides during a meal. Four groups of animals were used: rats that were food (but not water) deprived for 12-h period (preprandial group) and then given access to food for either 10 min (partially satiated group) or 35 min (postprandial group). The fourth group constituted nondeprived controls (ad libitum fed group). BN-like immunoreactivity (BLI) of the hypothalamus, hippocampus, and medulla was determined using a radioimmunoassay. Our data revealed that at the hypothalamus, the BLI content dropped significantly after food deprivation (preprandially), and returned to the ad libitum fed control levels after the meal (postprandially). At the hippocampus, food deprivation did not affect the BLI levels; however, food ingestion significantly elevated the BLI content within 35 min. The medullary BLI levels failed to alter in relation to the feeding status. The observed rapid alterations suggest that the hypothalamic response to food intake is satiety linked and hence lend support to the contention that BN-like peptides play a physiological role in the central regulation of ingestive behavior. The alterations noted at the hippocampus implicate physiological role of BN-like peptides in other meal-associated processes (such as memory).

Levels of immunoreactive insulin, neurotensin, and bombesin in porcine colostrum and milk.

High concentrations of insulin (411 +/- 214 microU/ml), neurotensin-like (265 +/- 72 pg/ml), and bombesin-like immunoreactivities (1995 +/- 288 pg/ml) were detected in porcine colostrum using radioimmunoassay, as compared to the levels found in sow blood serum at farrowing (5 microU/ml, less than 12 pg/ml, and 17 pg/ml, respectively). After 72 h of lactation, the levels of insulin and neurotensin-like immunoreactivities had decreased to 28 +/- 17 microU/ml and 89 +/- 23 pg/ml, respectively, while the bombesin-like activity remained constant. Characterization with reversed-phase high performance liquid chromatography showed that the insulin immunoreactivity eluted at the same position as the insulin standard, while the elution patterns of the neurotensin-like and bombesin-like immunoreactivities (eluted in three separate peaks) did not correspond to that of their respective standards. The biological function of the peptide hormones in colostrum/milk may be as triggers of the developmental changes taking place in the nursing neonate, especially in the gastrointestinal tract.

FMRFamide immunoreactivity in the nervous system of the medusa Polyorchis penicillatus.

Three different antisera to the molluscan neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and two different antisera to the fragment RFamide were used to stain sections or whole mounts of the hydrozoan medusa Polyorchis penicillatus. All antisera stained the same neuronal structures. Strong immunoreactivity was found in neurons of the ectodermal nerve nets of the manubrium and tentacles, in neurons of the sensory epithelium, and in neurons at the periphery of the sphincter muscle. Strong immunoreactivity was also present in processes and perikarya of the whole outer nerve ring, in the ocellar nerves, and in nerve cells lying at the periphery of the ocellus. The inner nerve ring contained a moderate number of immunoreactive processes and perikarya, which were distinct from the swimming motor neurons. In contrast to the situation in the hydrozoan polyp Hydra attenuata, no immunoreactivity was found with several antisera to oxytocin/vasopressin and bombesin/gastrin-releasing peptide. The morphology and location of most FMRFamide-immunoreactive neurons in Polyorchis coincides with two identified neuronal systems, which have been recently discovered from neurophysiological studies.

Comparative distribution of bombesin/GRP- and substance-P-like immunoreactivities in rat hypothalamus.

Immunohistochemical localization of bombesin/gastrin-releasing peptide ( GRP )-like immunoreactivity (BN/ GRP -LI) and substance P-like immunoreactivity (SP-LI) in consecutive sections of rat hypothalamus was studied. Bombesin/ GRP -like immunoreactivity in the hypothalamus was partially characterized by gel filtration chromatography followed by radioimmunoassay. In the hypothalamus, SP-LI was more widely distributed than BN/ GRP -LI. Only the anterior and medial parvocellular parts of the nucleus paraventricularis and the nucleus suprachiasmaticus contained numerous cell bodies which exhibited BN/ GRP -LI. Neurons in these areas did not exhibit SP-LI. In contrast, cell bodies exhibiting SP-LI were numerous in the nucleus preopticus medialis and lateralis, nucleus anterior, nucleus ventromedialis and dorsomedialis, nucleus lateralis, nucleus arcuatus, and nucleus premamillaris ventralis and dorsalis. Only occasional cell bodies in these areas exhibited BN/ GRP -LI. It is concluded that the neuronal systems in the hypothalamus containing BN/ GRP -LI and SP-LI are separate, though the terminal fields in many areas overlap. Two peaks of BN/ GRP -LI were detected after gel filtration chromatography from extracts of the rat nucleus paraventricularis. The high molecular weight form coeluted with synthetic GRP (1-27), and the small molecular weight form eluted after synthetic bombesin. Thus, the endogenous BN/ GRP -LI is probably not authentic bombesin.

Binding of GRP(14-27) but not bombesin or GRP(1-27) to hypothalamic magnocellular elements: an immunohistochemical study.

Bombesin, gastrin-releasing peptide (1-27), and gastrin-releasing peptide (14-27) abolished the specific immunocytochemical staining revealed by antiserum directed to the C-terminus of gastrin releasing peptide (GRP) and bombesin (BN) in rat hypothalamus. When the antiserum was preabsorbed with GRP(14-27), a strong reaction appeared in hypothalamic magnocellular neurons. This staining of magnocellular elements was produced by lower concentrations of GRP(14-27) than were needed to block immunocytochemical staining revealed by the antiserum in other hypothalamic locations. The distribution of GRP(14-27)-induced immunostaining was similar to that of neurophysin. Since only GRP(14-27) but not GRP(1-27) or bombesin was found to bind to magnocellular cells, it was concluded that binding was due to the N-terminus of GRP(14-27), which resembles the structure of oxytocin and vasopressin. In agreement with this, oxytocin and vasopressin were found to prevent the binding of GRP(14-27) to magnocellular cells. The similarity in localization and the effect of oxytocin and vasopressin suggest that GRP(14-27) may bind to neurophysin at low concentrations. The results suggest that enhancement of staining after preabsorption of antisera with antigens must be interpreted with care. Enhancement can occur at antigen concentrations lower than those required to block the immunostaining. These results fail to support the premise that antigen-induced enhancement of staining is due to antigen binding to specific receptors and subsequent detection of the receptor-bound antigen with the antiserum.

Cholecystokinin, bombesin and neurotensin in brain tissue from obese animals.

The concentration of cholecystokinin (the octapeptide, CCK-8), bombesin, and neurotensin was measured by radioimmunoassay in the cortex, hypothalamus and diencephalon of brains from lean, genetically obese and hypothalamic (VMH) obese rodents. Highest concentration of CCK-8 was found in the cortex whereas highest concentrations of bombesin and neurotensin were in the hypothalamus. When food was provided ad libitum, there was no difference in concentration of any of these peptides between lean and the respective genetically obese mice (ob/ob) and fatty (fa/fa) rats, or between lean and hypothalamic (VMH lesioned) obese rats. Adrenalectomy, which arrested the progression of obesity in both ob/ob and fatty rats, did not result in significant change in concentration of any of the three peptides studied in comparison with the respective sham-operated animals. Though significant differences in cholecystokinin and bombesin concentrations were detectable in some instances between adrenalectomized lean and adrenalectomized obese rats, these differences did not appear to be related to fall in food intake or slowing of body weight gain. Thus a variety of manipulations which altered the nutritional plane of the experimental rodents was not accompanied by significant changes in brain concentrations of cholecystokinin, bombesin or neurotensin.

24-hour variation in content and release of hypothalamic neuropeptides in the rat.

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.