Identification and characterization of growth hormone receptors in snakehead fish (Ophiocephalus argus cantor) liver.

The specific binding of 125I-labeled fish growth hormone (GH) to hepatic membranes prepared from several freshwater fish was assessed. A high level of growth hormone receptor (GHR) was detected on the hepatic membranes of the snakehead fish (Ophiocephalus argus Cantor). Scatchard analysis of the binding data showed a single class of high affinity binding site with a binding affinity (Ka) of 1.45 +/- 0.23 x 10(9) M-1 and a binding capacity (Bmax) of 198 +/- 57 fmol/mg protein. The binding was specific for fish GH and was saturable. In addition, the specific binding was temperature- and time-dependent, reaching a steady state after 16 hr of incubation at 25 degrees . The molecular weight of GHR as measured by Sephadex G-200 column chromatography and Western blot analysis using a monoclonal antibody (Mab263) against GHR was found to be 200-400 and 90-93 kDa, respectively. Two bands at 65 and 89 kDa were identified in ligand crosslinking studies of membrane receptors. A sensitive teleost GH radioreceptor assay (RRA) was developed, using recombinant fish GH and a membrane preparation from snakehead fish liver, capable of measuring bioactive GH in fish sera or other samples.

Quantitation of N-terminally extended tachykinins in cerebrospinal fluid from healthy subjects.

N-terminally extended substance P (SP) and neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), were determined in cerebrospinal fluid (CSF) from healthy human subjects by combined high performance liquid chromatography and radioimmunoassay. The concentrations of the peptides were similar in fresh CSF and in CSF which had been kept frozen for up to 5 months. SP and NKA were not present in measurable amounts in neither fresh CSF nor in CSF that had been frozen. On the other hand, when synthetic SP and NKA were added to approx. 2 pM concentration to fresh CSF samples, both peptides were recovered to 85 and 98%, respectively. There were no significant concentration gradients of the peptides in the first 18 ml (three consecutive 6 ml fractions) of CSF (n = 10). In contrast, we confirmed previous findings, that there are gradients of the amine metabolites 5-HIAA (P < 0.01) and HVA (P < 0.001) (n = 5). The concentrations of extended SP (expressed in SP equivalents) and NPK in the first 6 ml of CSF were 1.5 +/- 0.7 pM and 14.2 +/- 6.4 pM (mean +/- S.D., n = 10), respectively. The present results thus show that the levels of N-terminally extended SP and NKA are stable in frozen CSF samples for up to 5 months. The virtual lack of SP and NKA in CSF does not seem to be due to losses during sample preparation or storage.

N-terminally extended tachykinins in human cerebrospinal fluid.

With HPLC-RIA methods we were unable to demonstrate SP and NKA in measurable amounts in human CSF. Instead N-terminally extended forms of these peptides were found to be present and could be quantitated. The finding that these forms of tachykinins can be released from nervous tissue might suggest that their levels in CSF can be used as markers of the activity in central SP and NKA neurons.

Treatment of depression with E-10-hydroxynortriptyline--a pilot study on biochemical effects and pharmacokinetics.

The major metabolite of nortriptyline, i.e. E-10-hydroxynortriptyline (E-10-OH-NT), was given as a racemate in increasing doses from 75 to 225 mg/day to five patients with major depressive episode. Plasma concentrations of both the (-)- and (+)-enantiomers were linearly related to the doses. The mean ratio between them was 3.6 +/- 0.53, indicating stereospecific kinetics during maintenance treatment. Lumbar punctures were performed in four of the patients before and after 3 weeks of E-10-OH-NT treatment. There was a 18% mean decrease (P less than 0.01) in the noradrenaline metabolite HMPG in cerebrospinal fluid (CSF), supporting previous in vitro data showing that E-10-OH-NT inhibits noradrenaline uptake in vivo. During treatment, the median depression score measured by the Montgomery-Asberg Depression Rating Scale declined from 32 to 14 (P less than 0.05). As the study was open, the clinical outcome is not conclusive but does not contradict the hypothesis that E-10-OH-NT has antidepressant properties. If present at all, side effects were mild and did not interfere with the treatment.

N-terminally extended substance P is released together with substance P from rat spinal cord.

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.

Neuropeptide K is present in human cerebrospinal fluid.

Neurokinin A-like immunoreactivity (NKA-LI) in human cerebrospinal fluid (CSF) was determined by radioimmuno assay (RIA) combined with high performance liquid chromatography (HPLC). The major immunoreactive component did not coelute with NKA, but coeluted with neuropeptide K (NPK), which contains the NKA sequence in its C-terminus. Trypsin treatment of this component from human CSF and of synthetic NPK, produced a substance which coeluted with NKA in the HPLC system. When the NKA-LI was oxidized with hydrogen peroxide and rechromatographed, the immunoreactivity coeluted with NPK sulfoxide. The results indicate that the main part of the NKA-LI in CSF is identical with NPK. The mean concentration of NPK measured in CSF from 6 healthy subjects by HPLC-RIA was 23 +/- 11 (SD) pmol/L.

Fluoxetine treatment of depression. Clinical effects, drug concentrations and monoamine metabolites and N-terminally extended substance P in cerebrospinal fluid.

In an open study of depressed inpatients, the effects of the selective serotonin uptake blocker fluoxetine on 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenyl glycol (HMPG) and N-terminally extended substance P (SP) in cerebrospinal fluid (CSF) were measured. Thirteen unmedicated patients who met the DSM-III criteria for major depressive episode were included, and 9 completed the study. During treatment the 5-HIAA concentration decreased by 46%. The HVA and HMPG concentrations also decreased significantly, but to a lesser degree. The mean level of N-terminally extended SP was unaffected by fluoxetine treatment, but the pretreatment level correlated significantly with the pretreatment level of HMPG. The pretreatment level of HVA was the only biochemically variable that appeared to predict therapeutic outcome. The plasma concentrations of both fluoxetine and its metabolite norfluoxetine increased significantly between 3 and 6 weeks. Plasma and CSF levels of both the parent drug and its active metabolite were correlated.

Detection of N-terminally extended substance P but not of substance P in human cerebrospinal fluid: quantitation with HPLC-radioimmunoassay.

A reversed-phase HPLC system was used to concentrate and separate components of substance P-like immunoreactivity (SP-LI) from human CSF. When CSF was injected and fractions collected, no SP-LI could be detected by radioimmunoassay (RIA) at the retention time of SP or SP-sulfoxide. Instead, SP-LI was detected in later eluting fractions. This SP-LI reacted with two different antisera raised against the C-terminal part of SP, but not with an antiserum against the N-terminal part. A compound with similar properties was also found to be present in neutral extracts of rat dorsal spinal cord. When the late-eluting compound from human CSF was treated with trypsin and rechromatographed on HPLC, an immunoreactive component eluting at the position of SP could be detected with both the C- and N-terminally directed SP antisera. These results suggest that an N-terminally extended form of SP is present in human CSF. Trypsinization also gave two other compounds with affinity for the N- but not the C-terminally directed antisera. This may indicate that N-terminal fragments of SP extended at the N-terminus or SP molecules extended at both the N- and the C-terminus (i.e., preprotachykinins) also are present in human CSF. In 32 CSF samples from depressed patients, SP-LI was determined with a C-terminally directed antiserum with and without prior HPLC separation. SP itself could not be detected, but the late-eluting form of SP-LI could be quantitated in all samples by combined HPLC-RIA. In most samples, there was a relatively good agreement between the SP-LI levels measured with and without HPLC.

Immunoreactive vasopressin in bull seminal fluid.

Immunoreactive arginine vasopressin (irAVP) was measured in seminal fluid with and without extraction using a specific radioimmunoassay (RIA). A large fraction of irAVP was removed after extraction on octadecasilylsilica cartridges. The measured amount of irAVP corresponded to the levels found in blood plasma. Dilutions of seminal plasma extracts were parallel with the RIA standard curve. On reversed phase HPLC the extracted material coeluted with synthetic AVP. These findings suggest an identity of this immunoreactive material with intact AVP. During incubations of synthetic AVP and its analogue 8-D-arginine vasopressing (8-DAVP) in seminal plasma, immunoreactivity decreased considerably with the former peptide, while the concentration of 8-DAVP was not significantly altered.