Clinical hip tests and a functional squat test in patients with knee osteoarthritis: reliability, prevalence of positive test findings, and short-term response to hip mobilization.

STUDY DESIGN: One group pretest-posttest exploratory design. OBJECTIVES: Primary purposes of this study were to examine the short-term effect of hip mobilizations on pain and range of motion (ROM) measurements in patients with knee osteoarthritis (OA) and to determine the prevalence of painful hip and squat test findings in both patients with knee OA and asymptomatic subjects. The secondary purposes were to assess intrarater reliability and to determine whether fewer subjects experienced painful test findings following hip mobilization. BACKGROUND: Conservative intervention, including manual physical therapy applied to the lower extremity, has been shown to reduce impairments associated with knee OA. METHODS AND MEASURES: One rater pair administered 4 clinical hip tests to 22 patients with knee OA (mean age, 61.2 years; SD, 6.1 years) and 17 subjects without lower extremity symptoms or known pathology (mean age, 64.0 years; SD, 7.9 years). Intrarater reliability was examined for each clinical test. Patients with knee OA and painful-hip and squat test findings received hip mobilizations. Pain and ROM responses for each test were dependent variables. RESULTS: Intraclass correlation coefficients for all tests were greater than 0.87. Composite and individual test pain scores and ROM scores improved significantly following hip mobilization. All clinical test findings were more frequent in the group with knee OA, except for those of the FABER test, and the number of subjects with painful test findings following hip mobilization was reduced for all tests except the hip flexion test. CONCLUSIONS: Patients experienced increases in ROM, decreased pain, and fewer subjects had painful test findings immediately following a single session of hip mobilizations. Examination and intervention of the hip may be indicated in patients with knee OA.

The use of pigs as an animal model to evaluate the efficacy, potency and specificity of two growth hormone releasing factor analogues.

In 1982, Guillemin et al reported the isolation of the human (h) growth hormone (GH) releasing factor (GRF) from a pancreatic tumour in an acromegalic patient. Since then, work to develop potent GRF analogues has been widespread and the rat has been the main animal model used. The aim of the present study was to compare the efficacy, potency and specificity of two GRF analogues with those of the native GRF(1-29)NH(2)using pig (p) as the animal model. Two analogues, Al ([His(1), D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) and A2 ([D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) were compared with the h or pGRF(1-29)NH(2). Five studies were designed using 28-48 kg BW growing barrows. Results showed that the two GRF analogues were more potent than the native GRF molecule, were highly specific, were active for long periods of time and were able to induce changes in body composition similar to those reported with GH or other GRF analogues. Because of the similarity between swine and human species with respect to the amino acid sequence of GRF and to the physiology, secretion and effects of GH, it can be proposed that the pig could be used as a pre-clinical animal model to study and test new GRF molecules over short and long periods of time.

Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.

Antitumor and antiangiogenic effects of somatostatin receptor-targeted in situ radiation with (111)In-DTPA-JIC 2DL.

INTRODUCTION: Expression of somatostatin receptor subtype 2 (sst 2) in angiogenic tumor vessels appears to be homogeneous, while tumor cell expression of this receptor is often heterogeneous. We have developed a novel in vitro three-dimensional tumor angiogenesis model to study the antitumor and the antiangiogenic effects of radiolabeled somatostatin analogs. We hypothesized that targeted in situ radiation with an Auger electron-emitting radiolabeled somatostatin analog would produce receptor-specific cytotoxicity in sst 2-expressing cells. MATERIALS AND METHODS: IMR-32 human neuroblastoma (sst 2-positive) and MDA MB-231 human breast cancer (sst 2-negative) xenografts were created in nude mice from monolayer cell cultures. Fragments of these tumors were embedded in three-dimensional fibrin gels supplemented with endothelial growth media and incubated for a period of 14 days. Tumor fragments were treated with 50 microCi/ml of (111)In-JIC 2DL, a sst 2-preferring somatostatin analog, or medium on Day 1. Initial angiogenic activity was determined at 48 h and the mean angiogenic score and tumoricidal responses were assessed on Day 14. RESULTS AND CONCLUSION: Tumoricidal effects of (111)In-JIC 2DL were seen only in sst 2-positive IMR-32 tumors. However, the angiogenic response was inhibited in both IMR-32 and MDA MB-231 tumors independent of the tumor cells' sst 2 status. Somatostatin receptor-mediated in situ radiation therapy has profound cytotoxic effects on angiogenic blood vessels and sst 2-expressing tumor cells.

An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-alpha activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for beta-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed during hyperinsulinemic glucose clamps in TNFi obese animals, with no differences observed between the two lean groups. The improvement in peripheral and hepatic sensitivity to insulin seen in the obese animals was independent of insulin receptor (IR) number and insulin binding affinity for IR. However, TNF-alpha neutralization led to a 2.5-fold increase in tyrosine phosphorylation of IR in skeletal muscle, whereas this was unchanged in liver. There was also a 4-fold increase in particulate protein tyrosine phosphatase activity of skeletal muscle in TNFi obese animals vs. beta-galactosidase controls, whereas protein tyrosine phosphatase activity in liver was unchanged. These results suggest that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. TNF-alpha may affect insulin action in the liver either at sites distal to the IR or indirectly, possibly because of increased provision of gluconeogenic substrates or altered counterregulation. In addition, the Ad5-mediated gene delivery system employed here provides an in vivo model that is efficient and economical for exploring mechanisms involved in TNF-alpha-induced insulin resistance in various genetic models of obesity-linked diabetes.

Beneficial hemodynamic and renal effects of adrenomedullin in an ovine model of heart failure.

BACKGROUND: Adrenomedullin is a recently discovered endogenous peptide with hypotensive and natriuretic actions in normal animals. Circulating and ventricular adrenomedullin are elevated in congestive heart failure, suggesting a possible role in the pathophysiology of this disease. No studies have previously examined the effects of adrenomedullin in heart failure. METHODS AND RESULTS: Eight sheep with pacing-induced heart failure received human adrenomedullin(1-52) at 10 and 100 ng x kg(-1) x min(-1) I.V. for 90 minutes each. Compared with vehicle control data, adrenomedullin increased plasma cAMP (high dose, P<.05) in association with dose-dependent falls in calculated peripheral resistance (13 mm Hg x L(-1) x min(-1), P<.001), mean arterial pressure (9 mm Hg, P<.001), and left atrial pressure (5 mm Hg, P<.001) and increases in cardiac output (0.5 L/min, P<.001). Adrenomedullin increased urine sodium (threefold, P<.05), creatinine (P<.05) and cAMP excretion (P<.01), creatinine clearance (P<.05), and renal production of cAMP (P<.05), whereas urine output was maintained during infusion and raised after infusion (P<.05). Adrenomedullin reduced plasma aldosterone levels (P<.05), whereas plasma atrial and brain natriuretic peptide concentrations were unchanged during infusion and rose after infusion (P<.01 and P<.05, respectively). Plasma catecholamine, cortisol, renin, calcium, and glucose concentrations were not significantly altered. CONCLUSIONS: Adrenomedullin reduced ventricular preload and afterload and improved cardiac output in sheep with congestive heart failure. Despite the clear fall in arterial pressure, adrenomedullin increased creatinine clearance and sodium excretion and maintained urine output. These results imply an important pathophysiological role for adrenomedullin in the regulation of pressure and volume in heart failure and raise the possibility of a new therapeutic approach to this disease.

Tone-dependent vasodilator responses to proadrenomedullin NH2-terminal 20 peptide in the hindquarters vascular bed of the rat.

Responses to proadrenomedullin NH2-terminal 20 peptide (PAMP) were investigated in the systemic and hindquarters vascular bed of the rat. Intravenous injections of PAMP and adrenomedullin (ADM) produced dose-related decreases in systemic arterial and hindquarters perfusion pressure, which were not altered by alpha-receptor or adrenergic nerve terminal blocking agents. PAMP was 100-fold less potent than ADM, and hindquarters vasodilator responses to both peptides were similar in innervated and denervated preparations. When baseline tone was increased with phenylephrine and U46619 or decreased with sodium nitroprusside, vasodilator responses to PAMP and ADM were correlated with the basal level of tone, suggesting that responses to both peptides are dependent on the baseline level of vasoconstrictor tone in the hindquarters vascular bed of the rat.

In vivo evidence for progesterone dependent decreases in serotonin release in the hypothalamus and midbrain central grey: relation to the induction of lordosis.

The effects of progesterone (P) on serotonin (5HT) overflow in the ventromedial hypothalamus (VMH), preoptic area (POA) and midbrain central grey (MCG) were studied using in vivo microdialysis. Ovariectomized rats, pretreated with 5 micrograms estradiol, were anesthetized with chloral hydrate and stereotaxically implanted with dialysis probes directed towards one of the respective brain sites. Extracellular 5HT levels stabilized 3 to 5 h following probe implantation. Under stable baseline conditions, perfusion of 1 microM tetrodotoxin through the dialysis probe resulted in 60-65% reduction in 5HT overflow in the brain areas studied. In experiments testing the effect of P on 5HT overflow, rats were subcutaneously injected with 0.5 mg P or propylene glycol vehicle. Samples were analyzed for 5HT at 20 min intervals for 4 h after treatment. Perfusate levels of 5HT were not significantly changed in the VMH, POA or MCG in vehicle-treated rats. Similarly, P treatment failed to significantly alter 5HT overflow in the POA. In the VMH, perfusate levels of 5HT were significantly reduced 60 min after P treatment. Decreases in perfusate 5HT levels were detected 20 min after P in the MCG. The decreases in 5HT overflow measured in the VMH and MCG following P treatment persisted for the remainder of the sampling period with the exception of 1 time point in the VMH. The results provide in vivo evidence for P-influenced decreases in 5HT release in the VMH and MCG. The rapid decrease in extracellular 5HT in the MCG suggests that this effect may represent a non-genomic action of P. These results are discussed in relation to the role of 5HT in the regulation of lordosis behavior.

The somatostatin receptor subtype on rat enterochromaffinlike cells.

BACKGROUND/AIMS: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells. METHODS: ECL cells were isolated from rat fundic mucosa to a purity of 90%-95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture. RESULTS: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32-87, inhibited the gastrin-induced calcium signal at 10(-11) mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32-92, and also somatostatin 14 required 100-1000 times higher concentrations (10(-8) mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC50) value of 2 x 10(-12) mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC50 values of 1 to 5 x 10(-9) mol/L. CONCLUSIONS: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.

Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP.

Sensitive and specific two-side enzyme immunoassays (two-site EIAs) for pituitary adenylate cyclase activating polypeptides, PACAP38, and PACAP27, have been established using six monoclonal antibodies against PACAP38, and a rabbit antibody against a C-terminal portion of PACAP27. In extracts of rat hypothalamus, these EIAs detected not only PACAP38 and PACAP27 but also an immunoreactive (ir-) PACAP lacking an epitope of a monoclonal antibody, PA-1C, which recognizes the C-terminal portion of PACAP38. By the use of these EIAs, it was found that one of the human neuroblastoma cell lines, IMR-32, produced ir-PACAP. In reverse-phase (RP-)HPLC, intracellular and extracellular ir-PACAPs were separated into two peaks, of which one was eluted at a position close to that of PACAP38 and the other in rather hydrophobic fractions. Those ir-PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot analysis of the two peaks of the RP-HPLC indicated that they consisted of several components including those with apparent molecular weights of 6.5 k and 10 k for the first peak ir-PACAP, and 14 k and 20 k for the second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processing steps. Although the significance of the modification in the C-terminus of PACAP38 is unknown, IMR-32 may be a cell line useful for studying the regulation of the biosynthesis of PACAP.

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.

Effect of cyclosporine on steroidogenesis in rat Leydig cells.

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.

Effects of cyclosporine on the hypothalamic-pituitary-gonadal axis in the male rat: mechanism of action.

In the intact adult male rat, cyclosporine (CsA) induces a significant decrease in serum testosterone (T), serum LH, and intratesticular T. To elucidate the mechanism of action of this CsA-induced hypogonadotropic hypogonadism, castrated male rats were treated with oral CsA (30 mg/kg.day) or vehicle alone, and serum LH was measured after 1, 2, and 4 weeks of treatment. Surprisingly, serum LH was higher in these CsA-treated castrated rats at 1, 2, and 4 weeks than in castrated controls. To provide insight into the cause of this increase in serum LH, a T implant (8 mm) was inserted (at week 5 of treatment) in both CsA-treated and control castrated animals, and serum LH was measured 1 week after insertion of the T implant. Serum LH levels decreased to control values after insertion of the T implant. Subsequently, two other groups of rats received 8-mm T implant at the time of castration and were then treated up to 4 weeks with either CsA or vehicle alone. Serum LH showed a significant decrease in these CsA- plus T-treated animals compared to the vehicle- plus T-treated animals. A GnRH stimulation test performed after 2 weeks of CsA/vehicle treatment showed a significant increase in serum LH in all the rats 30 min after GnRH administration (1, 10, 30, and 100 ng/100 g BW, ip), indicating a normal pituitary response. The increase in LH after exogenous GnRH treatment was more significant in CsA-treated intact rats than in the controls. There was no difference in creatinine clearance between intact and castrate T-treated rats regardless of whether they received CsA. These studies indicate that the hypogonadotropic hypogonadism induced by CsA in intact male rats is mediated through the hypothalamic-pituitary axis, primarily at its hypothalamus end, does not seem to be due to the nephrotoxicity of CsA, and is modulated by T and/or its metabolites.

Structure-activity studies of antagonists of luteinizing hormone-releasing hormone with emphasis on the amino-terminal region.

The structure-activity relationship of the hydrophobic amino terminal region of the antagonist [N-Ac-D-Nal1,D-pClPhe2,D-Trp3,D-Arg6,Phe7,D-Ala10]-LH- RH has been investigated by the incorporation of a variety of amino acids with emphasis on positions 1, 2, and 3. The analogues were prepared by routine solid-phase peptide synthesis. All purifications were performed in two stages: gel permeation chromatography followed by preparative, reversed-phase, high-performance chromatography. The analogues were assayed in a standard rat antiovulatory assay using a 40% propane-1,2-diol-saline vehicle. A simplified antagonist was developed that allowed the removal of the custom-synthesized D-pClPhe and the labile D-Trp while retaining antiovulatory potency. The compound [N-Ac-D-Nal1,D-Phe2,3,D-Arg6,Phe7,D-Ala10]-LH-RH caused a 56% blockade of ovulation at the 500-ng dose and is approximately equipotent with the parent analogue in this system.

Cranial irradiation for cerebral and nasopharyngeal tumours in children: evidence for the production of a hypothalamic defect in growth hormone release.

A synthetic 29-amino acid analogue of human pancreatic GH-releasing hormone (GHRH(1-29)NH2) has recently been shown to stimulate the release of GH in normal subjects. We have studied the GH response to GHRH(1-29)NH2 in nine children irradiated for brain and nasopharyngeal tumours, who were not growing and were deficient in GH as assessed by insulin-induced hypoglycaemia. Serum GH rose in response to GHRH(1-29)NH2 in all the children, and in five the peak serum GH response was greater than 20 mu./l. The data suggest that when hypothalamo-pituitary irradiation results in GH deficiency, this is due to a failure of the synthesis or delivery of endogenous GHRH from the hypothalamus to the pituitary cells. It also suggests that it may be possible to treat such children using synthetic GHRH in place of exogenous GH.

Growth hormone releasing factor: comparison of two analogues and demonstration of hypothalamic defect in growth hormone release after radiotherapy.

Human pancreatic growth hormone releasing factor (hpGHRF(1-40] stimulates the release of growth hormone in normal subjects and some patients with growth hormone deficiency. A study comparing the shorter chain amidated analogue hpGHRF(1-29) with an equivalent dose of hpGHRF(1-40) in seven normal subjects showed no significant difference in growth hormone response between the two preparations. Six patients with prolactinomas were also tested; these patients had received megavoltage radiotherapy previously but had developed growth hormone deficiency as shown by insulin induced hypoglycaemia. In all six patients 200 micrograms hpGHRF(1-40) or hpGHRF(1-29)NH2 produced an increase in the serum growth hormone concentration. These data suggest that hpGHRF(1-29)NH2 may be useful for testing the readily releasable pool of growth hormone in the pituitary and that cases of hypothalamo-pituitary irradiation resulting in growth hormone deficiency may be due to failure of synthesis or delivery of endogenous GHRF from the hypothalamus to pituitary cells.

Effects of H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH on the in vitro uptake and release of radiolabelled dopamine, noradrenaline and serotonin in rat brain hypothalamic slices.

The effects of H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH, a hypothalamic heptapeptide with weak CRF activity were investigated on the in vitro uptake and release of radiolabelled dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in rat brain hypothalamic slices. In a doses of 5 X 10(-6) M and 10(-5) M the heptapeptide significantly increased the 5-HT uptake, whereas it had no effect on the DA and NA uptakes. The same doses (5 X 10(-6) M, 10(-5) M) significantly increased the DA release without affecting the NA and 5-HT release. These results suggest that H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH as a hypothalamic peptide is able to modulate selectively the activity of the serotonergic and dopaminergic transmission without influencing the noradrenergic system in the hypothalamus.

Growth-hormone-releasing factor in growth hormone deficiency: demonstration of a hypothalamic defect in growth hormone release.

Four patients with hypothalamic tumours or idiopathic growth hormone (GH) deficiency, who were GH deficient by conventional criteria, responded to 200 micrograms synthetic hpGRF-40 with a clear rise in circulating GH.

Characterization of binding sites for N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in rat brain.

Binding sites for N-Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1), a novel peptide structurally related but immunoreactively different from Pro-Leu-Gly-NH2 (MIF-1), were investigated. The presence of Tyr-MIF-1-like material in brain tissue has previously been demonstrated by RIA and its levels were shown to vary with the diurnal cycle and after pinealectomy. We now demonstrate a high affinity binding site in rat brain that is saturable and specific for Tyr-MIF-1. Crude P2 synaptosomal fractions from rat brains were incubated with 125I-Tyr-MIF-1 in the presence or absence of 10 microM unlabeled Tyr-MIF-1 (nonspecific binding). Binding reached equilibrium at 30-40 min at 23 degrees C and at about 4 hr on ice, after which it was relatively stable for at least 18 hr. None of the other peptides (including MIF-1) or amino acid residues tested were found to effectively compete for 125I-Tyr-MIF-1 binding. Binding was linear with protein from 280 micrograms to at least 1.1 mg protein per tube. Scatchard analysis of the striatum-thalamus revealed the presence of binding sites with an apparent Kp of 91 nM and maximum number of sites in the range of 45 fmol/mg tissue. Analysis of several brain areas revealed a differential distribution of the binding sites with relatively high concentrations in cortex, striatum, and amygdala and low concentrations in pons-medulla. Together with the previously published RIA results, the demonstration of a receptor for Tyr-Pro-Leu-Gly-NH2 supports the concept of the presence of this novel peptide and its receptor in the brain.

Radioimmunoassayable N-Tyr-MIF-1-like activity in rat brain is increased by pinealectomy.

Levels of N-Tyr-MIF-1-like immunoreactivity were measured in rat brain by radioimmunoassay (RIA). Although the highest levels were found in the pineal and hypothalamus, the striatum and thalamus also contained significantly more immunoreactivity than the other parts of the brain. Since oxytocin cross-reacts 5.4% with the antibody for N-Tyr-MIF-1, oxytocin-like immunoreactivity was also measured by RIA, but could not account for the levels of radioimmunoassayable N-Tyr-MIF-1 found in these experiments. There was a significant increase in N-Tyr-MIF-1-like immunoreactivity after pinealectomy, a tendency for a decrease after stress, but essentially no change after hypophysectomy. A diurnal rhythm was observed with the highest levels at night and lowest levels during the day. The results demonstrate the presence in brain tissue of a novel immunoreactive peptide, the levels of which can be altered by neuroendocrine manipulations.