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Knee osteoarthritis (OA) is a chronic degenerative inflammatory-based condition caused by a cascade of different intra-articular molecules including several cytokines. Among the cytokines, prokineticins (PKs) have recently been identified as important mediators of inflammation and pain. This observational study examined the potential involvement of PK2 in degenerative or traumatic knee disease. Fifteen patients presenting knee osteoarthritis (OA group) and 15 patients presenting a traumatic meniscal tear (TM group) were studied. Synovial fluid samples from affected knees were assessed for PK2, IL-10, and TNF-α using the ELISA method. At a long-term follow-up (minimum 5 years, mean = 6.1 years), patients in the TM group underwent clinical re-evaluation with PROMs (Tegner Activity Scale, IKDC, Lysholm, SKV); in addition, X-ray visualization was used to assess the presence of secondary OA. PK2 was detected in synovial fluids of both TM and OA patients and the levels were comparable between the two groups, while IL-10 levels were significantly greater in the OA group than those in TM patients. PK2 levels correlated with those of IL-10. PK2 levels were greater in blood effusions compared to clear samples, did not differ significantly between sexes, nor were they related to differences in weight, height, or injury (meniscal laterality, time since dosing). No correlation was found between PROMs and radiological classifications in patients in the TM group at final follow-up. These data are the first observations of PK2 in synovial fluid following traumatic meniscus injury. These findings suggest possible further prognostic indices and therapeutic targets to limit the development of secondary OA.

Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease whose etiology remains unresolved; nonetheless, mutations of superoxide dismutase 1 (SOD1) have been associated with several variants of ALS. Currently available pharmacologic interventions are only symptomatic and palliative in effect; therefore, there is a pressing demand for more effective drugs. This study examined potential therapeutic effects of growth hormone secretagogues (GHSs), a large family of synthetic compounds, as possible candidates for the treatment of ALS. Human neuroblastoma cells expressing the SOD1-G93A mutated protein (SH-SY5Y SOD1G93A cells) were incubated for 24 h with H2O2 (150 µM) in the absence, or presence, of GHS (1 µM), in order to study the protective effect of GHS against increased oxidative stress. The two GHSs examined in this study, hexarelin and JMV2894, protected cells from H2O2-induced cytotoxicity by activating molecules that regulate apoptosis and promote cell survival processes. These findings suggest the possibility of developing new GHS-based anti-oxidant and neuroprotective drugs with improved therapeutic potential. Further investigations are required for the following: (i) to clarify GHS molecular mechanisms of action, and (ii) to envisage the development of new GHSs that may be useful in ALS therapy.

Purpose: This study examined and compared all-suture anchors with metallic anchors in the arthroscopic repair of small to medium supraspinatus tears; a multi-dimensional evaluation protocol assessed (i) clinical, (ii) ultrasonographical, and (iii) functional criteria at mid-term. Materials and methods: A sample of 40 patients, prescreened from a larger group who had undergone arthroscopic rotator cuff repair, were resolved into two groups of 20, based on the type of anchor used in their surgery (all-suture or metallic) and were evaluated postoperatively (mean = 44.3 months, SEM = 22.4 months, minimum period = 10.3 months). Clinical assessments included (i) objective and subjective scores (Constant, DASH, SPADI, SST), (ii) ultrasonographical examination, as well as (iii) isometric and isokinetic strength testing. Raw data from the operated side was compared with those from the contralateral side within each group. Raw data for the PROMs and for the US evaluation were compared between groups. Strength data, as well as the Constant score values, were further analyzed based on (i) the difference between operated and intact sides, as well as (ii) the strength ratio between operated and intact sides; the transformed data were then compared between the two groups. Results: We found no statistically significant differences in any of the parameters considered between shoulders operated using ASAs or MAs, nor between operated and non-operated sides in each group. Conclusion: Our findings indicate that the reliability and performance characteristics of all-suture anchors and metallic anchors are practically indistinguishable for arthroscopic treatment of small to medium lesions of the supraspinatus tendon.

Introduction The incidence of anterior cruciate ligament (ACL) tears in skeletally immature patients has acutely increased over the last 20 years, yet there is no consensus on a single "best treatment". Selection of an optimal treatment is critical and based on individual circumstances; consequently, we propose a treatment-selection algorithm based on (i) skeletal development, (ii) ACL tear location, type, and quality, as well as (iii) parental perspective in order to facilitate the decision-making process. Evidence acquisition We combined our surgical group's extensive case histories of ACL tear management in Tanner Stage 1 and 2 patients with those in the literature to form a consolidated data base. For each case (i) the diagnostic phase, (ii) communication with patient and parents, (iii) treatment choice(s), (iv) selected surgical techniques and (v) rehabilitation schedule were critically analyzed and compared for patient outcomes. Evidence synthesis MRI-imaging and intraoperative tissue quality assessment were preeminent in importance for selection of the optimal treatment strategy. Considerations for selecting an optimal treatment included: (i) associated lesions, (ii) the child/patient and parent(s)' well-informed and counseled consent, (iii) biological potential, and (iv) the potential for successful ACL preservative surgery. Complete ACL tears were evaluated according to tear-location. In Type I and II ACL tears with remaining good tissue quality, we propose primary ACL repair. In Type III and IV ACL tears we propose physeal-sparing reconstruction with an iliotibial band graft. Finally, in the case of a Type V ACL tear, we propose that the best treatment be based on the Meyers-McKeever classification. Conclusions We present a facile decision-making algorithm for ACL management in pediatric patients based on specific elements of tissue damage and status.

Osteochondroma is one of the most common benign bone tumors; however, as it rarely affects any bones in feet, it may easily escape detection without rigorous examination. We present an exceptionally rare case of tarsal navicular dorsal osteochondroma diagnosed in an 11-year-old female child affected by chronic foot pain. Radiographs, MRI, and computed tomography scan revealed bony excrescences extending outward from the navicular bone. After conservative treatment failed, the navicular dorsal exostosis was excised in open surgery with complete resolution of symptoms. Navicular-cuneiform impingement was diagnosed by instrumental and intraoperative techniques. Histological analysis confirmed the diagnosis of navicular dorsal osteochondroma. This case report illustrates the necessity of particularly rigorous evaluation of the substrates of pediatric chronic foot pain.

Introduction: Surgical repair of the rotator cuff is based on the use of anchors whose ideal numbers and configurations continue to be controversial. We compared the clinical-functional results arising from the arthroscopic repair of shoulders, with small-medium lesions of the supraspinatus tendon, among patients using one anchor with three sutures, or two anchors with two sutures. Methods: In this retrospective study patient were resolved into 2 groups. Clinical and functional results were assessed based on Constant Score and instrumental isometric examination. Results: Patients in Group 1 experienced shoulder repair using a single anchor with three sutures (n = 21, mean age = 56 years, range = 51–65). In Group 2, patients received two anchors with two sutures each (n = 24, mean age = 59 years, range = 24–75). The mean follow-up time was 15 months. The mean values of the operated shoulders’ Constant Score were 88.05 and 88.25 respectively. Examination of isometric test results in operated shoulders, healthy shoulders and the two different rotator cuff repair techniques did not reveal any statistically significant differences. Conclusion: In the arthroscopic repair of small-medium supraspinatus tendon tears, the short to mid-term clinical and functional outcomes arising from use of 1 triple-loaded or 2 double-loaded metallic sutures anchors are comparable.

Hexarelin, a synthetic hexapeptide, exerts cyto-protective effects at the mitochondrial level in cardiac and skeletal muscles, both in vitro and in vivo, may also have important neuroprotective bioactivities. This study examined the inhibitory effects of hexarelin on hydrogen peroxide (H2O2)-induced apoptosis in Neuro-2A cells. Neuro-2A cells were treated for 24 h with various concentrations of H2O2 or with the combination of H2O2 and hexarelin following which cell viability and nitrite (NO2−) release were measured. Cell morphology was also documented throughout and changes arising were quantified using Image J skeleton and fractal analysis procedures. Apoptotic responses were evaluated by Real-Time PCR (caspase-3, caspase-7, Bax, and Bcl-2 mRNA levels) and Western Blot (cleaved caspase-3, cleaved caspase-7, MAPK, and Akt). Our results indicate that hexarelin effectively antagonized H2O2-induced damage to Neuro-2A cells thereby (i) improving cell viability, (ii) reducing NO2− release and (iii) restoring normal morphologies. Hexarelin treatment also reduced mRNA levels of caspase-3 and its activation, and modulated mRNA levels of the BCL-2 family. Moreover, hexarelin inhibited MAPKs phosphorylation and increased p-Akt protein expression. In conclusion, our results demonstrate neuroprotective and anti-apoptotic effects of hexarelin, suggesting that new analogues could be developed for their neuroprotective effects.

Purpose The aim of this study was to assess midterm clinical outcomes in Tanner 1–2 patients with proximal anterior cruciate ligament (ACL) tears following arthroscopic-surgical repair using an absorbable or an all-suture anchor. Methods Fourteen (9.2 ± 2.9 years-old) of 19 skeletally immature patients reached the 2 years of clinical follow-up. Physical examinations included the Lachman test, Pivot-shift test, One-leg Hop test, Pedi-IKDC as well as Lysholm and Tegner activity scores; knee stability was measured with a KT-1000 arthrometer. Overall re-rupture rates were also evaluated in all operated patients. Results At 2 years post-surgery, the Lysholm score was 93.6 ± 4.3 points, and the Pedi-IKDC score was 95.7 ± 0.1. All patients returned to the same sport activity level as prior to ACL lesion within 8.5 ± 2.9 months, with one exception who reported a one-point reduction in their Tegner Activity score. No leg-length discrepancies or malalignments were observed. Four patients presented grade 1 Lachman scores, and of these, three presented grade 1 (glide) score at Pivot-shift; clinical stability tests were negative for all other patients. Anterior tibial shift showed a mean side-to-side difference of 2.2 mm (range 1–3 mm). The One-leg Hop test showed lower limb symmetry (99.9% ± 9.5) with the contralateral side. Overall, 4 out of 19 patients presented a re-rupture of the ACL with a median time between surgery and re-rupture of 3.9 years (range 1–7). Conclusion This surgical technique efficiently repairs proximal ACL tears, leading to a restoration of knee stability and a quick return to an active lifestyle, avoiding growth plate disruption. Level of evidence IV.

Hexarelin, a synthetic hexapeptide, protects cardiac and skeletal muscles by inhibiting apoptosis, both in vitro and in vivo. Moreover, evidence suggests that hexarelin could have important neuroprotective bioactivity.Oxidative stress and the generation of free radicals has been implicated in the etiologies of several neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and multiple sclerosis. In addition to direct oxidative stress, exogenous hydrogen peroxide (H2O2) can penetrate biological membranes and enhance the formation of other reactive oxygen species.The aim of this study was to examine the inhibitory influence of hexarelin on H2O2-induced apoptosis in Neuro-2A cells, a mouse neuroblastoma cell line. Our results indicate that H2O2 reduced the viability of Neuro-2A cells in a dose-related fashion. Furthermore, H2O2 induced significant changes in the morphology of Neuro-2A cells, reflected in the formation of apoptotic cell bodies, and an increase of nitric oxide (NO) production. Hexarelin effectively antagonized H2O2 oxidative damage to Neuro-2A cells as indicated by improved cell viability, normal morphology and reduced nitrite (NO2−) release. Hexarelin treatment of Neuro-2A cells also reduced mRNA levels of caspases−3 and −7 and those of the pro-apoptotic molecule Bax; by contrast, hexarelin treatment increased anti-apoptotic Bcl-2 mRNA levels. Hexarelin also reduced MAPKs phosphorylation induced by H2O2 and concurrently increased p-Akt protein expression.In conclusion, our results identify several neuroprotective and anti-apoptotic effects of hexarelin. These properties suggest that further investigation of hexarelin as a neuroprotective agent in an investigational and therapeutic context are merited.

Background In children, fracture non-union is uncommon yet, curiously, non-union of distal fibula fractures are rarely reported. Historically, the most common treatment of a lateral malleolus fracture after an ankle sprain is conservative, which usually leads to fracture union. However, even in clinically stable ankles, subsequent pain arising from fracture site could suggest non-union, thereby necessitating reexamination and possible secondary treatment. Case presentation We report the case of an 8-year-old girl with an epiphyseal distal fibula fracture complicated with a symptomatic non-union associated with the chondral flap of the talar dome after conservative treatment. Surgical excision of the fragment and chondroplasty was performed and resulted in an excellent clinical outcome. Conclusion This case report illustrates the necessity of particularly meticulous evaluation of pediatric post-traumatic ankle pain. Surgical treatment as well as talar chondral evaluation should be taken into consideration in the treatment of pediatric distal fibular nonunion.

The specific etiology of meniscal tears, including the mechanism of lesion, location, and orientation, is considered for its contribution to subsequent joint cytokine responsiveness, healing outcomes, and by extension, appropriate lesion-specific surgical remediation. Meniscal repair is desirable to reduce the probability of development of posttraumatic osteoarthritis (PTOA) which is strongly influenced by the coordinate generation of pro- and anti-inflammatory cytokines by the injured cartilage. We now present biochemical data on variation in cytokine levels arising from two particular meniscal tears: bucket-handle (BH) and posterior horn (PH) isolated meniscal tears. We selected these two groups due to the different clinical presentations. We measured the concentrations of TNF-α, IL-1β, IL-6, IL-8, and IL-10 in knee synovial fluid of 45 patients with isolated meniscal lesions (BH tear, n = 12 ; PH tear, n = 33 ). TNF-α levels were significantly ( p < 0.05 ) greater in the BH group compared with the PH group, whereas IL-1β levels were significantly greater ( p < 0.05 ) in the PH group compared with the BH group. Both BH and PH groups were consistent in presenting a positive correlation between concentrations of IL-6 and IL-1β. A fundamental difference in IL-10 responsiveness between the two groups was noted; specifically, levels of IL-10 were positively correlated with IL-6 in the BH group, whereas in the PH group, levels of IL-10 were positively correlated with IL-1β. Collectively, our data suggest a possible influence of the meniscal tear pattern to the articular cytokine responsiveness. This differential expression of inflammatory cytokines may influence the risk of developing PTOA in the long term.

Neurodegenerative diseases, including Alzheimer disease (AD), Parkinson disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington disease, are characterized by the loss of neurons as well as neuronal function in multiple regions of the central and peripheral nervous systems. Several studies in animal models have shown that androgens have neuroprotective effects in the brain and stimulate axonal regeneration. The presence of neuronal androgen receptors in the peripheral and central nervous system suggests that androgen therapy might be useful in the treatment of neurodegenerative diseases. To illustrate, androgen therapy reduced inflammation, amyloid-β deposition, and cognitive impairment in patients with AD. As well, improvements in remyelination in MS have been reported; by comparison, only variable results are observed in androgen treatment of PD. In ALS, androgen administration stimulated motoneuron recovery from progressive damage and regenerated both axons and dendrites. Only a few clinical studies are available in human individuals despite the safety and low cost of androgen therapy. Clinical evaluations of the effects of androgen therapy on these devastating diseases using large populations of patients are strongly needed.

Introduction: This study evaluates mid-term results of acetabular revision using a hemispherical acetabular cup in Trabecular Titanium with a cage construct. Methods: We reviewed 36 acetabular revisions performed with the Delta Revision TT cup in 34 patients (mean age = 75, range: 45–92 years). Acetabular defect types (Paprosky classification) included (1) 2B ( n = 5), (2) 2C ( n = 7), (3) 3A ( n = 15), and (4) 3B ( n = 9). Morcellised bone allografts were used in 24 cases, and synthetic bone used in 11. Outcomes were evaluated using the Harris Hip Score (HHS), and the Verbal Rating Scale (VRS) for pain measurement. X-ray visualisation of cup position was used to discern signs of mobilisation and bone graft incorporation. Survivorship at post-revision follow-up (mean = 39.8; range 12–91.5 months) was calculated. Results: HHS increased from 40.5 to 87 ( p < 0.01). 68% of cases were pain free; by comparison, 32% had an average VRS score of 1.9 (range 1–3). The average cup inclination angle was 40.8° (30–52°) postoperatively, compared with 41.2° (30–52°) at follow-up; there were no signs of loosening or mobilisation. Centre of rotation was fully restored in 21 (58.3%) hips. According to Gie classification; bone graft incorporation grades were (1) 3 ( n = 21), (2) 2 ( n = 12) and (3) 1 ( n = 2). The survival rate was 100% for aseptic loosening and 91.7% for any cause of revision. Conclusions: The Delta Revision TT cup promises good clinical and radiographic results at short- to mid-term follow-up, with high rates of survival rate and bone integration.

Androgens in women, as well as in men, are intrinsic to maintenance of (i) reproductive competency, (ii) cardiac health, (iii) appropriate bone remodeling and mass retention, (iii) muscle tone and mass, and (iv) brain function, in part, through their mitigation of neurodegenerative disease effects. In recognition of the pluripotency of endogenous androgens, exogenous androgens, and selected congeners, have been prescribed off-label for several decades to treat low libido and sexual dysfunction in menopausal women, as well as, to improve physical performance. However, long-term safety and efficacy of androgen administration has yet to be fully elucidated. Side effects often observed include (i) hirsutism, (ii) acne, (iii) deepening of the voice, and (iv) weight gain but are associated most frequently with supra-physiological doses. By contrast, short-term clinical trials suggest that the use of low-dose testosterone therapy in women appears to be effective, safe and economical. There are, however, few clinical studies, which have focused on effects of androgen therapy on pre- and post-menopausal women; moreover, androgen mechanisms of action have not yet been thoroughly explained in these subjects. This review considers clinical effects of androgens on women's health in order to prevent chronic diseases and reduce cancer risk in gynecological tissues.

TLQP-21, a peptide encoded by the prohormone VGF, is expressed in neuroendocrine cells and can modulate inflammatory processes. Since TLQP-21 can bind the complement 3a receptor 1 on macrophages, interest has risen in this peptide as a potential drug for the treatment of Acute Respiratory Distress Syndrome (ARDS), whose hospital mortality can reach 35–46%. Since no effective pharmacologic therapies are available, our aim was to exploit the potential of a short analog of TLQP-21(JMV5656) in order to modulate the inflammatory process in ARDS and the progression to pulmonary fibrosis in an experimental model of unilateral acid aspiration in mice. Mice were divided in 2 treatment groups. In the acute protocol, mice received intra-peritoneal injection of either vehicle or 0.6 mg/kg JMV5656 on experimental days 1 and 2, and ARDS was induced on day 3 under deep anesthesia by instillation of HCl (1.5 ml/kg of 0.1 M HCl in 0.9% NaCl) into the right lung; all measurements were performed 24 h later. In the subacute protocol, mice were treated as previously, but treatment with vehicle or JMV5656 was repeated also on day 4 and measurements were made 2 weeks later. Twenty-four hours after acid instillation, the total number of immune cell in the BAL rose sharply due primarily to an increase in the PMN population which increased from 1% up to 58% of total cell numbers. JMV5656 significantly reduced PMN recruitment into the alveolar space, but had no effects on cytokine levels in BAL. Two weeks after acid injury, static compliance of the right lung was significantly higher in the JMV5656-treated group compared to vehicle-treated group. Treatment with JMV5656 also blunted the acid-induced collagen deposition in the right lung. These results suggest that JMV5656 can ameliorate mechanical compliance, and reduce collagen deposition in acid-injured lungs in mice. This effect was likely due to the ability of JMV5656 to inhibit PMN recruitment in the injured lung.

Introduction: Ideal treatment of acute acromioclavicular joint (ACJ) dislocation remains unresolved. We evaluated ACJ reconstruction using polyester tapes and temporary Kirschner wire (KW) and presented clinical and radiological outcomes. Materials and methods: Patients were retrospectively evaluated and classified according to Rockwood classification, clinical and sportive characteristics. Constant Score (CS) and ACJ joint instability (ACJI) score were collected. Zanca’s, Alexander, axillary, standard, and stress AP views were collected. Radiographic coracoclavicular distance (CCd) of healthy and injured shoulders was measured pre-operatively, at 3months and at minimum 2-year follow-up. Heterotopic ossifications and clavicular osteolysis were evaluated. Influence of patients’ characteristics and AC joint type on clinical and radiological outcomes were determined. Results: Sixteen patients (13 type V and 3 type III ACJ dislocation) reached the end of follow-up (mean 2.4years; range 22–72months). Mean CS was 99.63 (range 96–100), while the mean ACJI score was 96.19 (range 85–100). The CCd of the treated shoulder was comparable with the healthy shoulder at 3months and at last follow-up; moreover, there was no difference in CCd scores at 3months and at 2years. CCd scores were lower in sedentary patients compared with physically active (sporty) patients. Heterotopic ossifications were observed in three patients. No osteolysis was reported. Conclusion: This technique provides good results with few complications and should be considered as an effective method to treat ACJ acute dislocation. CCd scores correlate with overuse factors but not with other clinical scores.

Background: Metabolic complications represent a common and serious problem associated with HIV infection and combined Antiretroviral Therapy (cART). Alterations in body fat distribution are associated with significantly increased risks of (i) metabolic derangements, (ii) cardiovascular pathologies, and (iii) insulin resistance. A case control study showed that in subcutaneous adipose tissue from HIV-infected patients on cART presenting lipodystrophy (LS), the levels of miRNA-218 were upregulated and those of lipin-1, a putative target gene of miRNA-218, were downregulated compared with HIV-negative subjects. Lipin-1 is one of the most important factors linked to development of LS. Lipin-1, by controlling PPARγ2, regulates the expression of specific genes, such as that of glucose transporter type 4 (GLUT-4), required for maturation and maintenance of adipocytes. Objectives: To determine whether lopinavir/ritonavir (LPV/RTV) can modulate lipogenesis in adipocytes affecting miRNA-218 and lipin-1 mRNA expression, and to investigate the functional link between miRNA-218 and GLUT-4 mRNA expression. Methods: Differentiated 3T3-L1 cells were treated with various combinations of LPV/RTV, followed by measurements of cell viability, lipid accumulation, lipin-1 and GLUT-4 mRNA and miRNA-218 levels. Transfection of anti-miR-218 or a miRNA-218 mimic were used to investigate the role of miRNA-218 in lipogenesis. Results: LPV/RTV treatment of 3T3-L1 cells did not affect the viability of differentiated 3T3-L1 cells, but caused (i) a significant decrease of lipid accumulation, (ii) an overexpression of miRNA-218, and (iii) a reduction of lipin-1 and GLUT-4 mRNA levels. The anti-miR-218 transfection of 3T3-L1 cells significantly ameliorated the adipogenic dysfunction and restored mRNA levels of lipin-1 and GLUT-4 consequent to LPV/RTV treatment. By contrast, 3T3-L1 cells transfected with a specific miRNA-218 mimic showed (i) an overexpression of miRNA-218, (ii) a reduced cellular lipid fraction, and (iii) decreased levels of mRNA for lipin-1 and GLUT-4. Conclusion: 3T3-L1 cells, treated with LPV/RTV, show altered lipid content due to increased miRNA-218 levels, which affects lipin-1 mRNA. Moreover, increased miRNA-218 levels were inversely correlated with changes in GLUT-4 expression, which suggests a role for miRNA-218 in mediating the insulin resistance consequent to cART.

Concentrations of pro- and anti-inflammatory cytokines in synovial fluid samples collected from patients with chronic meniscal tears were investigated. An acute inflammatory response is generally reported 24-48 h after knee injury, but the largest body of data available in literature concerns anterior cruciate ligament injury and very little information is available about the balance of soluble factors in the synovial fluid of knees with chronic meniscal tears. Sixty-nine patients (46 males and 23 females) with meniscal tear that occurred more than 3 months earlier were enrolled. According to cartilage integrity assessment by arthroscopic examination, patients were assigned to one of the following groups: (i) no chondral damage (n = 18); (ii) chondral damage graded from I to II (n = 15); and (iii) chondral damage graded from III to IV (n = 37). In all groups, levels of IL-10 and inflammatory cytokines IL-6, TNF-α, and IL-8 where greater compared with those reported in the intact population; by contrast, levels of IL-1ra and IL-1β were significantly lower. Interestingly, IL-6 levels were higher in female than male patients. Cytokine levels did not correlate with degree of chondral damage. IL-6 and IL-1ra levels positively correlated with IL-1β, and negatively correlated with TNF-α. Interestingly, levels of IL-1β and TNF-α were inversely correlated. Our data demonstrate increased levels of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α) in the chronic phase of meniscal trauma. This pro-inflammatory state is maintained in the joint from the time of initial injury to several months later and could be a key factor in hampering cartilage regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:340-346, 2017.

TLQP-21 is a neuropeptide which has been implicated in regulation of nociception and other relevant physiologic functions. Although recent studies identified C3a and gC1q receptors as targets for TLQP-21, its intracellular molecular mechanisms of action remain largely unidentified. Our aim was (i) to explore the intracellular signaling pathway(s) activated by JMV5656, a novel derivative of TLQP-21, in RAW264.7 macrophages, and (ii) to assess linkages of these pathways with its purported receptors. JMV5656 stimulated, in a dose-dependent fashion, a rapid and transient increase in intracellular Ca2+ concentrations in RAW264.7 cells; repeated exposure to the peptide resulted in a lower response, suggesting a possible desensitization mechanism of the receptor. In particular, JMV5656 increased cytoplasmic Ca2+ levels by a PLC-dependent release of Ca2+ from the endoplasmic reticulum. STIM proteins and Orai Ca2+ channels were activated and played a crucial role. In fact, treatment of the cells with U73122 and thapsigargin modulated the increase of intracellular Ca2+ levels stimulated by JMV5656. Moreover, in RAW264.7 cells intracellular Ca2+ increases did not occur through the binding of JMV5656 to the C3a receptor, since the increase of intracellular Ca2+ levels induced by JMV5656 was not affected by specific siRNA against C3aR. In summary, our study provides new indications for the downstream effects of JMV5656 in macrophages, suggesting that it could activate receptors different from the C3aR.

The treatment of anterior cruciate ligament (ACL) injuries in children and adolescents is challenging. Preclinical and clinical studies investigated ACL repairing techniques in skeletally immature subjects. However, intra-articular bioenvironment following ACL tear has not yet been defined in skeletally immature patients. The aim of this study was to measure cytokine concentrations in the synovial fluid in adolescent population. Synovial levels of IL-1β, IL-1ra, IL-6, IL-8, IL-10, and TNF-α were measured in 17 adolescent patients (15 boys) with ACL tears who underwent ACL reconstruction including acute (5), subacute (7), and chronic (5) phases. Femoral growth plates were classified as “open” in three patients, “closing” in eight, and “closed” in six. Eleven patients presented an ACL tear associated with a meniscal tear. The mean Tegner and Lysholm scores (mean±SD) of all patients were 8 ± 1 and 50.76 ± 26, respectively. IL-8, TNF-α, and IL-1β levels were significantly greater in patients with “open” physes. IL-1ra and IL-1β levels were significantly higher in patients with ACL tear associated with a meniscal tear. Poor Lysholm scores were associated with elevated IL-6 and IL-10 levels. IL-10 levels positively correlated with IL-6 and IL-8 levels, whereas TNF-α concentration negatively correlated with IL-6 levels. Skeletally immature patients with meniscal tears and open growth plates have a characteristic cytokine profile with particularly elevated levels of proinflammatory cytokines including IL-8, TNF-α, and IL-1β. This picture suggests that the ACL tear could promote an intra-articular catabolic response in adolescent patients greater than that generally reported for adult subjects. The study lacks the comparison with synovial samples from healthy skeletally immature knees due to ethical reasons. Overall, these data contribute to a better knowledge of adolescent intra-articular bioenvironment following ACL injuries.

International audience; VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervous system. VGF and peptides derived from its processing have been found in dense core vesicles and are released from neuronal and neuroendocrine cells via the regulated secretory pathway. Among VGF-derived neuropeptides, TLQP-21 (VGF556-576) has raised a huge interest and is one of most studied. TLQP-21 is a multifunctional neuropeptide involved in the control of several physiological functions, potentially including energy homeostasis, pain modulation, stress responsiveness and reproduction. Although little information is available about its receptor and the intracellular mechanisms mediating its biological effects, recent reports suggest that TLQP-21 may bind to the complement receptors C3aR1 and/or gC1qR. The first aim of this study was to ascertain the existence and nature of TLQP-21 binding sites in CHO cells. Secondly, we endeavored to characterize the ligand binding to these sites by using a small panel of VGF-derived peptides. And finally, we investigated the influence of TLQP-21 on selected intracellular signaling pathways. We report that CHO cells express a single class of saturable and specific binding sites for TLQP-21 with an affinity and capacity of Kd = 0.55 ± 0.05 × 10-9 M and Bmax = 81.7 ± 3.9 fmol/mg protein, respectively. Among the many bioactive products derived from the C-terminal region of VGF that we tested, TLQP-21 was the most potent in stimulating intracellular calcium mobilization in CHO cells; this effect is primarily due to its C-terminal fragment (HFHH-10). TLQP-21 induced rapid and transient dephosphorylation of phospholipase Cγ1 and phospholipase A2. Generation of IP3 and diacylglycerol was crucial for TLQP-21 bioactivity. In conclusion, our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, and its activation first increases membrane-lipid derived second messengers which thereby induce the mobilization of Ca2+ from the endoplasmic reticulum followed by a slower store-operated Ca2+ entry from outside the cell.

International audience; TLQP-21 (TLQPPASSRRRHFHHALPPAR) is a multifunctional peptide that is involved in the control of physiological functions, including feeding, reproduction, stress responsiveness, and general homeostasis. Despite the huge interest in TLQP-21 biological activity, very little is known about its intracellular mechanisms of action. In microglial cells, TLQP-21 stimulates increases of intracellular Ca2+ that may activate functions, including proliferation, migration, phagocytosis and production of inflammatory molecules. Our aim was to investigate whether JMV5656 (RRRHFHHALPPAR), a novel short analogue of TLQP-21, stimulates intracellular Ca2+ in the N9 microglia cells, and whether this Ca2+ elevation is coupled with the activation Ca2+-sensitive K+ channels. TLQP-21 and JMV5656 induced a sharp, dose-dependent increment in intracellular calcium. In 77% of cells, JMV5656 also caused an increase in the total outward currents, which was blunted by TEA (tetraethyl ammonium chloride), a non-selective blocker of voltage-dependent and Ca2+-activated potassium (K+) channels. Moreover, the effects of ion channel blockers charybdotoxin and iberiotoxin, suggested that multiple calcium-activated K+ channel types drove the outward current stimulated by JMV5656. Additionally, inhibition of JMV5656-stimulated outward currents by NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4 benzothiazin-3(4H)-one) and TRAM-34 (triarylmethane-34), indicated that KCa3.1 channels are involved in this JMV5656 mechanisms of action. In summary, we demonstrate that, in N9 microglia cells, the interaction of JMV5656 with the TLQP-21 receptors induced an increase in intracellular Ca2+, and, following extracellular Ca2+ entry, the opening of KCa3.1 channels.

Oncologic patients subjected to chemotherapy frequently present aphagia, malnutrition, and cachexia. The purpose of this study was to investigate whether selected growth hormone secretagogues including hexarelin, JMV2894 and JMV2951 could antagonize body weight loss and wasting induced by cisplatin administration in rats. The three growth hormone secretagogues behaved as full agonists of the growth hormone secretagogues receptor both in terms of ability to stimulate calcium mobilization in Chinese hamster ovary cells and stimulation of growth hormone release in neonatal rats. Adult rats were (i) treated with vehicle throughout (controls), or (ii) treated with cisplatin (days 1–3) and a growth hormone secretagogues or vehicle, (days 1–12). Body weight and food consumption were measured daily. Although all growth hormone secretagogues caused initial transient acute increases in food intake, the total amount of food eaten by controls and growth hormone secretagogues treated groups over the 12 experimental days was not significantly different. All groups pre-treated with cisplatin lost up to 5–10 % body weight in the first 4 days; they subsequently gained weight at a rate comparable with controls. Interestingly, rats which received JMV2894 demonstrated a faster gain in body weight than any other growth hormone secretagogues treated group and at the end of the protocol reached a weight similar to that of controls. JMV2894 did not stimulate perirenal and epididymal fat accumulation but reduced MuRF mRNA levels in skeletal muscles. In conclusion, our findings demonstrate that JMV2894 antagonizes cisplatin induced weight loss in rats and may prove useful in antagonizing cachexia associated with cancer and chemotherapy in humans.

Anterior cruciate ligament (ACL) reconstruction restores knee stability but does not reduce the incidence of posttraumatic osteoarthritis induced by inflammatory cytokines. The aim of this research was to longitudinally measure IL-1β, IL-6, IL-8, IL-10, and TNF-αlevels in patients subjected to ACL reconstruction using bone-patellar tendon-bone graft. Synovial fluid was collected within 24–72 hours of ACL rupture (acute), 1 month after injury immediately prior to surgery (presurgery), and 1 month thereafter (postsurgery). For comparison, a “control” group consisted of individuals presenting chronic ACL tears. Our results indicate that levels of IL-6, IL-8, and IL-10 vary significantly over time in reconstruction patients. In theacute phase,the levels of these cytokines in reconstruction patients were significantly greater than those in controls. In thepresurgeryphase, cytokine levels in reconstruction patients were reduced and comparable with those in controls. Finally, cytokine levels increased again with respect to control group in thepostsurgeryphase. The levels of IL-1βand TNF-αshowed no temporal variation. Our data show that the history of an ACL injury, including trauma and reconstruction, has a significant impact on levels of IL-6, IL-8, and IL-10 in synovial fluid but does not affect levels of TNF-αand IL-1β.

Surgical reconstruction of the anterior cruciate ligament (ACL) does not necessarily decrease the risk of developing osteoarthritis (OA). The inflammatory response and relative changes in pro- and anti-inflammatory cytokines could participate in triggering the development of OA. To test this hypothesis we measured the concentrations of IL-1β, IL-1ra, IL-6, IL-8, IL-10, and TNF-α at different times after ACL rupture. The sample population consisted of 48 patients with ACL tear which were assigned to different groups according to the time elapsed from the injury: 22 acute (A), 7 early sub-acute (ESA), 11 late sub-acute (LSA), and 8 chronic (C). In group A, there were high levels of IL-1β, IL-6, and IL-8, whereas levels of IL-1ra and TNF-α were significantly lower than usually reported. IL-1β and IL-8 concentrations returned with time to normal levels in the ESA group. Interestingly, IL-1ra levels remained always significantly lower than normally reported levels, and TNF-α levels did not increase after trauma. Our data show increased level of pro-inflammatory cytokines (IL-6 and IL-8) in the acute phase of inflammation which could be responsible for triggering cartilage catabolism and suggest that prompt neutralization of IL-6 and IL-8 accumulations in synovial fluid could help prevent development of OA in ACL-injured knees.

This study compared the distribution of specifically bound [3H]ketanserin (Bsp) with serotonin (5HT) in brain regions of juvenile and sexually recrudescing female trout. Amounts of Bsp varied widely among brain regions and consistently differed between juvenile and sexually recrudescing females. Levels of Bsp were significantly greater in the hypothalamus than the olfactory lobe, which were at least threefold greater than in all other tissues examined (Kruskal-Wallis test, p < 0.05). Bsp densities in the hypothalamus, preoptic area, and optic lobe were significantly greater in juveniles compared with corresponding tissues from sexually recrudescing females (Mann-Whitney U test, p < 0.05); in contrast, Bsp in olfactory lobe and spinal cord did not differ significantly between the two classes of fish. 5HT concentration was determined by high performance liquid chromatography - electrochemical detection (HPLC-EC) analysis. Biogenic amine standards eluted in a stereotypic pattern, with peaks consistently separable in time. 5HT concentration was significantly greater in hypothalamus than in olfactory lobe and undetectable in the pituitary (Kruskal-Wallis test, p < 0.05). Trends in distribution of Bsp and 5HT were comparable in the hypothalamus and preoptic area in juvenile and sexually recrudescing females. In general, density of specific [3H]ketanserin binding sites was directly related to 5HT content of brain regions in juvenile and sexually recrudescing females. 5HT concentrations (pmol/g tissue) were approximately 900-fold greater than Bsp (fmol/g tissue) in all brain regions, and approximately 300-fold greater than Bsp in the olfactory lobe. These results suggest important regulatory role(s) for 5HT in the trout preoptic-hypothalamo-hypophysial axis, which may differ from 5HT role(s) in trout olfactory lobe.Key words: high performance liquid chromatography - electrochemical detection, [3H]ketanserin, sexually recrudescing female trout.

This study examines the existence and pharmacological specificity of [3H]ketanserin binding in hypothalamus of juvenile rainbow trout. Hypothalamic membranes were incubated with [3H]ketanserin (selective 5HT2 antagonist) under several experimental conditions; reactions were terminated by filtration and bound radioactivity was counted by liquid scintillation spectroscopy. Tissue dilution experiments revealed that specific [3H]ketanserin binding (Bsp) was tissue dependent; 1 hypothalamus equivalent per tube (1100 ± 115 cpm/mg protein) was subsequently used throughout the rest of this study. In association experiments, Bsp increased progressively with time, achieved equilibrium binding levels (1192 ± 120 cpm/mg protein) within 80 min, and remained stable for at least 60 min thereafter; kobs, and k+1 were 0.032 and 0.048 min-1·nM-1, respectively. In dissociation experiments, Bsp completely dissociated within 20 min following addition of excess ketanserin; k-1 and t1/2 were 0.0803 min-1 and 8.7 min, respective...

This study examines the existence and pharmacological specificity of [3H]ketanserin binding in hypothalamus of juvenile rainbow trout. Hypothalamic membranes were incubated with [3H]ketanserin (selective 5HT2 antagonist) under several experimental conditions; reactions were terminated by filtration and bound radioactivity was counted by liquid scintillation spectroscopy. Tissue dilution experiments revealed that specific [3H]ketanserin binding (Bsp) was tissue dependent; 1 hypothalamus equivalent per tube (1100 ± 115 cpm/mg protein) was subsequently used throughout the rest of this study. In association experiments, Bsp increased progressively with time, achieved equilibrium binding levels (1192 ± 120 cpm/mg protein) within 80 min, and remained stable for at least 60 min thereafter; kobs, and k+1 were 0.032 and 0.048 min-1·nM-1, respectively. In dissociation experiments, Bsp completely dissociated within 20 min following addition of excess ketanserin; k-1 and t1/2 were 0.0803 min-1 and 8.7 min, respectively. Bsp was saturable (2500 ± 256 cpm/mg protein); Scatchard-calculated values for the equilibrium dissociation constant (KD) and capacity (Bmax) were 0.48 nM, and 125 fmol/mg protein, respectively. Bsp was differentially displaced by structurally related competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > serotonin (5HT) = spiperone >> methiothepin mesylate > metergoline = DOI ((±)-2-5-dimethoxy-4-iodoamphetamine hyrobromide) > 2-methyl-5HT > alpha-methyl-5HT >>>> 5HIAA (5-hydroxyindole acetic acid) = reserpine. These findings provide pharmacological evidence for the presence of a 5HT2-like receptor subtype in the trout hypothalamus.Key words: hypothalamus, [3H]ketanserin, 5HT2 receptor subtype, rainbow trout.

Indirect evidence suggests that glucocorticoid hormones may act through cellular receptors to play a neuromodulatory role in the teleost CNS. We now report our findings on the use of [3H]dexamethasone (DEX) to identify hypothalamic glucocorticoid receptors (GRs) in juvenile rainbow trout, Oncorhynchus mykiss. Hypothalamic cytosol was incubated with [3H]DEX under various experimental paradigms with incubations terminated by addition of dextran-coated charcoal; following immediate centrifugation, a sample of bound [3H]DEX (supernatant) was collected and assessed for 3 H content. [3H]DEX binding was tissue dependent between 0.5 and 2. 0 hypothalamus equivalents per tube (1.0 to 4.7 mg protein, respectively). Specific binding (BSP) increased with time for 1.5 h and remained relatively constant for an additional 2.5 h; the calculated association rate constant was 2.23 x 10(8) M-1 x min-1. Equilibrium BSP was dissociated by addition of a 5000 M excess cortisol with an accompaning t1/2 of 1.25 h and dissociation rate constant of 0.553 min-1. BSP was saturable with a calculated equilibrium Kd and BMAX of 1.22 nM and 296 fmol/mg protein, respectively. BSP was displaced under equilibrium conditions by the corticosteroids, but to a lesser extent by the mineralocorticoid, estrogen, and progestin. The rank order of potency for [3H]DEX displacement was DEXcortisolcorticosteronem triamcinolone = 11-deoxycortisolaldosteroneprogesterone17 beta-estradiol. These properties of specifically bound [3H]DEX indicate the presence of a GR, similar to the mammalian cytosolic GR, in the hypothalamus of juvenile rainbow trout.

Surgical reconstruction of the anterior cruciate ligament (ACL) does not necessarily decrease the risk of developing osteoarthritis (OA). The inflammatory response and relative changes in pro- and anti-inflammatory cytokines could participate in triggering the development of OA. To test this hypothesis we measured the concentrations of IL-1β, IL-1ra, IL-6, IL-8, IL-10, and TNF-α at different times after ACL rupture. The sample population consisted of 48 patients with ACL tear which were assigned to different groups according to the time elapsed from the injury: 22 acute (A), 7 early sub-acute (ESA), 11 late sub-acute (LSA), and 8 chronic (C). In group A, there were high levels of IL-1β, IL-6, and IL-8, whereas levels of IL-1ra and TNF-α were significantly lower than usually reported. IL-1β and IL-8 concentrations returned with time to normal levels in the ESA group. Interestingly, IL-1ra levels remained always significantly lower than normally reported levels, and TNF-α levels did not increase after trauma. Our data show increased level of pro-inflammatory cytokines (IL-6 and IL-8) in the acute phase of inflammation which could be responsible for triggering cartilage catabolism and suggest that prompt neutralization of IL-6 and IL-8 accumulations in synovial fluid could help prevent development of OA in ACL-injured knees. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 315-321, 2013 Copyright © 2012 Orthopaedic Research Society.

Dopamine, a catecholamine neurohormone, modulates pituitary hormone release in teleost fishes and other vertebrates. The existence and binding parameters of a pituitary dopamine–neuroleptic receptor from trout were examined and compared with those from goldfish. Pituitary homogenate was incubated with [3H]spiperone (D2 antagonist) under several experimental paradigms; incubations were terminated by filtration and bound 3H radioactivity was assessed by liquid scintillation spectroscopy. Specific binding of [3H]spiperone was tissue dependent. Equilibrium displacement analyses using domperidone (D2 antagonist) indicated a single class of binding site (LIGAND) with Kd = 2.49 ± 0.89 μM and a capacity of 3.10 ± 0.45 nmol/mg protein; the goldfish Kd and capacity were both significantly (p d = 4.63 ± 0.30 μM and capacity = 20.66 ± 2.03 nmol/mg protein. The Kd and capacity for the trout pars distalis (2.45 ± 0.33 μM and 3.27 ± 0.24 nmol/mg protein, respectively) did not differ significantly (p 2 receptor ligands differentially displaced [3H]spiperone from the trout pituitary, while D1 ligands, a D4 ligand, and a 5-hydroxytryptamine (5HT2) receptor antagonist had only small nonspecific effects. Comparison of the trout and goldfish pituitary dopamine–neuroleptic receptor indicates conservation of receptor affinity (Kd); however, differences in receptor numbers and in the distribution of receptors between the pars distalis and neurointermediate lobe in the two species may be due in part to species or developmental differences, and may reflect differences in the role(s) and degrees of influence of dopamine in these fishes.Key words: pituitary, dopamine, receptor, rainbow trout, goldfish.

The mechanisms of cardiovascular protective effects of ghrelin and its synthetic analogs are still largely unknown. Our first aim was to ascertain whether or not natural and synthetic ligands of GHS-R1a are capable of interfering with the activity of the renin-angiotensin system. Second, since polymorphisms in the ACE gene have been associated with Alzheimer's dementia (AD) and ACE is potentially involved in brain β-amyloid degradation, we also investigated the state of ghrelin axis and inflammatory markers in patients with AD and vascular dementia (VaD). Desacyl ghrelin, hexarelin, EP80317, and GHRP-6 all significantly inhibited ACE activity in vitro; by comparison, the efficacies of ghrelin and MK-0677 were significantly lower, suggesting that ACE-inhibiting activity is unrelated to ligand affinity to GHS-R1a. ACE was capable of cleaving Aβin vitro, reducing its ability to aggregate in fibrillar Aβ. Interestingly, this protective effect of ACE was blunted by enalapril but not hexarelin or EP80317. Desacyl ghrelin levels were lower in VaD subjects compared with AD and control subjects, whereas ghrelin and TNF-α levels were similar in all groups. VaD subjects demonstrated greater levels of mRNA for GHS-R1a, PPAR-γ and CD36 in peripheral blood lymphocytes compared with other groups. In conclusion, some GHSs are effective ACE-inhibitors, and this activity may contribute to their cardiovascular effects. Hexarelin or EP80317 do not inhibit the N-domain of ACE, which is also involved in the metabolism of β-amyloid, suggesting the possibility of developing new antihypertensive drugs with improved therapeutic potential.

Lewis (LEW/N) and Fischer (F344/N) rats represent two extremes of the spectrum of corticosterone responses to stressful stimuli, from the chronical hyporesponsiveness of LEW/N to the chronical hyperresponsiveness of F344/N. It might be expected that the amount of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) binding, and the levels of their corresponding mRNAs in various tissues in LEW/N and F344/N rats might reflect the overall integrated levels of corticosterone to which these receptors have been exposed. We have found that while the binding affinity (Kd) of MR and GR varies between tissues, there was no strain difference in any tissue. Receptor binding number (Bmax), however, varied not only between tissues, but also between strains. MR Bmax in the hippocampus and pituitary was lower in LEW/N than in F344/N, whereas the GR Bmax in the LEW/N thymus was greater than that found in F344/N rats. The hippocampal levels of MR mRNAs in Adx LEW/N and F344/N rats were in good agreement with, and paralleled, the functional levels of these receptors as determined by binding assays. On the other hand, the number of hippocampal GR binding sites and the level of GR nRNA while similar were not identical in the two strains: the hippocampal GR Bmax did not differ between strains, while the hippocampal GR mRNA level was slightly, but significantly, lower in Adx LEW/N compared to F344/N rats.

The gonadotropin secretory response of anterior pituitary cells to phorbol esters includes both extracellular Ca2+-dependent and -independent components ( Stojilkovicet al, 1988; J. Biol. Chem. 263, 17301–17306, 1988 ). In cultured pituitary cells, measurements of [Ca2+]i using Fura-2 and of LH release during cell perifusion studies revealed that the initial effects of phorbols and permeant diacylglycerols on these responses are extracellular Ca2+-dependent and are mediated through activation of voltage- and dihydropyridine-sensitive calcium channels. On the other hand, pretreatment with phorbol esters for 30 to 60 min inhibited subsequent [Ca2+]i responses to diacylglycerols and phorbols and significantly reduced agonist-induced biphasic [Ca2+]i responses, with no change in the number of GnRH receptors. These findings demonstrate that protein kinase C exerts both positive and negative control of [Ca2+]i, and indicate that the calcium, phospholipid dependent enzyme participates in the activation of voltage-sensitive calcium channels and hormone secretion in pituitary gonadotrophs.

Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [3H]17beta-estradiol (E2) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B(SP)) of [3H]E2 was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B(SP) in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B(SP) in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k(+1)) for cytosol and nuclear extract preparations were 1.10 +/- 0.02 x 10(8) M(-1) min(-1) and 1.27 +/- 0.04 x 10(8) M(-1) min(-1), respectively. Equilibrium bound B(SP) dissociated from cytosol preparations with a half life (t1-2) of 42 min and a dissociation rate constant (k(-1)) of 1.01 +/- 0.03 min(-1). B(SP) dissociated from nuclear extract preparations with a t1-2 = 45 min and k(-1)= 0.92 +/- 0.01 min(-1) x B(SP) was saturable in both extract preparations with a calculated equilibrium dissociation constant (Kd) of 1.46 +/- 0.1 nM (cytosol) and 2.37 +/- 0.2 nM (nuclear), and a maximum number of binding sites (B(MAX)) of 50.85 +/- 3.2 fmol mg(-1) protein and 61.74 +/- 2.65 fmol mg(-1) protein, respectively. In both preparations, B(SP) was differentially displaced by structurally similar compounds with a rank order of potency of E2estroneestriol17alpha-ethynyl estradioltestosteroneprogesterone = tamoxifencortisoldexamethasonebeta-sitosterol. These properties of specifically bound [3H]E2 suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.

Binding parameters of radiolabeled pGlu-3-Me-His2-Pro-NH2 ([3H]MeTRH) to pituitary TRH receptors of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were investigated. Washed pituitary membranes were incubated with [3H]MeTRH in the absence (B0) or presence of TRH or TRH analogs under various paradigms. Specific binding (Bsp) was thermolabile and tissue dependent. Association of Bsp was slow (k + 1 = 1.43 x 10(7) M-1 min-1) achieving equilibrium binding after 60 min, and remaining stable for at least 60 min. Thereafter, equilibrium-bound [3H]MeTRH was dissociated by addition of excess MeTRH; estimated dissociation rate constant (k - 1) and half-life (t1/2) were 6.74 x 10(-2) min-1 and 10.2 min, respectively, with a kinetically derived dissociation constant (k - 1/k + 1) of 4.71 x 10(-9) M. [3H]MeTRH binding was differentially displaced by TRH and TRH analogs. LIGAND analysis of multiple homologous (MeTRH) and heterologous (TRH) displacement experiments indicates the presence of a single class of binding sites with nanomolar affinity. Data pooled from multiple heterologous (TRH) displacement experiments suggested the presence of an additional class of higher-affinity (picomolar) binding sites. Additionally, examination of separate preparations of neurointermediate lobes and pars distalis from trout and goldfish suggested that these two classes of binding sites were differentially located between the two lobes. Specificity analysis of the trout pituitary TRH receptor indicates that the rank order of potency for [3H]MeTRH displacement was MeTRH > TRH > pGlu-Phe-Pro-NH2. Analogs with N- or C-terminal substitutions had little competitive potential. This study indicates the presence and binding characteristics of specific TRH receptors in the salmonid and cyprinid pituitary gland.

Seasonal variations in the serum concentrations of gonadotropin (GtH) and the serum GtH response to intraperitoneal injection of domperidone, a specific dopamine receptor antagonist, were examined in goldfish. In addition, the effects of in vivo treatment of goldfish with a superactive analog of salmon gonadotropin-releasing hormone (sGnRH-A) and domperidone on the binding parameters of pituitary GnRH and dopamine receptors were investigated in goldfish. Serum concentrations of GtH and the maximum GtH response to domperidone increased in correlation with advancing gonadal maturation; values increased from those in sexually regressed fish in January to maximal levels observed in fish in late stages of gonadal recrudescence in March, followed by a decrease with gonadal regression. At all stages, injection of domperidone increased serum concentrations of GtH in a dose-related manner; however, the ED50 of domperidone did not vary significantly over the course of the reproductive cycle. Multiple injections of sGnRH-A caused a progressively increasing and more prolonged serum GtH response; as well, multiple sGnRH-A treatment significantly potentiated the serum GtH response to domperidone without altering the ED50 of domperidone. sGnRH-A treatment caused a significant increase in the number of dopamine/neuroleptic receptors in the goldfish pars distalis, accompanied by a nonsignificant increase in dopamine/neuroleptic receptors in the neurointermediate lobe, and significantly increased the number of high-affinity GnRH receptors in the goldfish pituitary. Treatment with domperidone also significantly increased pituitary high-affinity GnRH receptor numbers. Receptor affinities were not significantly altered by either sGnRH-A or domperidone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Homogenates of the pituitary of the goldfish ( Carassius auratus ) were incubated with [ 3 H]spiperone under various experimental paradigms to evaluate the binding characteristics of the goldfish pituitary dopamine receptor. Binding was tissue specific as binding of [ 3 H]spiperone to goldfish pituitary was greater than other tissue types examined, and the magnitude of binding was found to be dependent on pituitary (protein) content; also, specific binding was heat labile. Association was rapid and binding was reversible (dissociable) by addition of excess competing ligand (domperidone, a specific dopamine D2 receptor antagonist); the half-life ( t 1 2 ) of dissociation was 9.2 min and the estimated dissociation rate constant ( k −1 ) was 7.56 × 10 −2 min −1 ); as well, the association rate was temperature dependent. Binding was saturable; saturation analysis using [ 3 H]spiperone indicated a single class of binding sites with an estimated dissociation constant ( K d ) and capacity of 7.39 ± 1.23 × 10 −6 M and 31.56 ± 2.72 × 10 −9 mol/mg protein, respectively. [ 3 H]Spiperone binding was displaceable; displacement analysis using unlabeled domperidone indicated a single class of binding sites with estimated K d and capacity of 2.94 ± 0.54 × 10 −6 M and 19.47 ± 3.12 × 10 −9 mol/mg protein, respectively. Binding was specifically inhibited by various dopamine antagonists and agonists. The density of binding sites differed significantly between regions of the goldfish pituitary; the number of binding sites in the pars distalis and neurointermediate lobes was estimated as 38.89 ± 2.07 × 10 −9 mol/mg protein vs 109.45 ± 25.33 × 10 −9 mol/mg protein, respectively; while the K d 's estimated as 3.73 ± 0.248 × 10 −6 M vs 4.1 ± 1.21 × 10 −6 M , respectively, were not significantly different. These data agree with previous in vivo and in vitro findings of the biological actions of dopamine agonists and antagonists in modifying gonadotropic hormone release in the goldfish and represent the first demonstration of the existence and binding characteristics of a dopamine/neuroleptic receptor in the pituitary of nonmammalian vertebrate.

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.