Acute effects of morphine on the expression of mRNAs for NMDA receptor subunits in the rat hippocampus, hypothalamus and spinal cord.

The acute effects of subcutaneously injected morphine on transcripts of the NMDA receptor subunits NR1, NR2A and NR2B in certain areas of the central nervous system of male rats were examined by Northern blot analysis. The result clearly indicated that a single dose (10 mg/kg) of the opioid alters the expression of the mRNA for receptor subunits in the hippocampus and hypothalamus 4 h after drug injection. No change in the mRNA levels was observed 30 min following injection, and after 24 h most of the levels were restored to control values. The observation suggests that morphine affects this type of glutamate receptor already in the acute phase of its administration.

Elevated cerebrospinal fluid level of substance P and decreased undecapeptidase activity at term pregnancy.

This paper reports a study of substance P and its converting enzyme substance P endopeptidase (SPE) in cerebrospinal fluid (CSF) collected from women at term pregnancy. A method was developed to assess the CSF levels of substance P itself with minimum contribution from prestages or fragments of the undecapetide. The measured activity was compared with that detected in CSF from control, non-pregnant, non-puerperal women. The result indicates a significant increase in substance P-like immunoreactivity at term pregnancy (19.9 +/- 3.9 fmol/ml, n = 10) compared with that of control subjects (12.3 +/- 2.8 fmol/ml, n = 9; P < 0.001). This elevation was suggested to reflect an increased activity in spinal sensory neurons at this stage of pregnancy. The activity of SPE was assessed by measuring the product substance P1-7 by a radioimmunoassay specific for this fragment. It was found that the enzyme activity was significantly decreased in CSF from pregnant women (11.2 +/- 0.71 pmol/h.ml), compared with control samples (18.4 +/- 0.73 pmol/h.ml; P < 0.05). Moreover, a significant negative correlation was found to exist between the level of substance P and the activity of SPE (P < 0.05, r2 = 0.65), suggesting that the enzyme may be involved in a mechanism regulating the level of substance P concentration.

Effects of an anabolic-androgenic steroid on the regulation of the NMDA receptor NR1, NR2A and NR2B subunit mRNAs in brain regions of the male rat.

The expression of the N-methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A and NR2B mRNAs was examined in discrete areas of the male rat brain (including hippocampus, hypothalamus, nucleus accumbens and cortex) following 14 days daily intramuscular injections of high doses (5 and 15 mg/kg) of an anabolic-androgenic steroid (AAS) (nandrolone decanoate). The results indicated that the drug produced a significant decrease in the mRNA expression of the NR2A receptor subunit both in the hypothalamus and hippocampus. A decrease in the level of NR2B receptor mRNA was observed in hypothalamus at the lower dose of the AAS but in other areas examined, this receptor subunit mRNA was not affected. Except for a decreased expression in the nucleus accumbens at the higher dose of AAS the NR1 receptor subunit mRNA was not affected by the drug. The three subunit mRNAs in cortex were not significantly altered. The effects of the steroid on the mRNA expression for the NMDA receptor subunits in hippocampus and hypothalamus are suggested to be involved in the mechanism behind aggressive behaviour, a feature previously associated with AAS misuse. The downregulation of the mRNA for the NR1 receptor subunit in nucleus accumbens may relate to a mechanism involved in the recently suggested AAS-induced stimulation of the brain reward system.

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Immunohistochemical detection of the melanocortin 1 receptor in human testis, ovary and placenta using specific monoclonal antibody.

We describe the immunohistochemical detection of the melanocortin 1 receptor (MC1R) protein in human gonadal tissues using a specific monoclonal antibody. The MC1R was found to be present in Leydig's cells in testis, in lutein cells in the corpus luteum and in the nucleus of the trophoblastic cells of the placenta. Though it has been speculated earlier that MC1R is present in gonadal tissues, this is the first report demonstrating the presence of MC1R protein in these cells.

Substance P endopeptidase activity in the rat spinal cord following injury: influence of the new anti-oxidant compound H 290/51.

The influence of the new antioxidant compound H-290/51 was examined on the substance P endopeptidase (SPE) activity in a rat model of spinal cord injury. This compound (H-290/51) has neuro-protective effects on edema and cell changes in this model. Infliction of trauma to the cord by making an incision into the right dorsal horn of the T10-11 segment resulted in a marked upregulation of SPE in the segments rostral to the lesion. On the other hand, the injured and adjacent caudal segments exhibited a marked down-regulation of the enzyme activity. Pretreatment with H 290/51 increased the SPE activity in the T9 segment but downregulated the enzyme activity in the T10-11 and T12 segments. The drug induced enzyme activity change was not further influenced by the trauma of the cord. The results indicate that a focal trauma induces widespread alterations in spinal cord SPE activity which can be influenced by the anti-oxidant drug H 290/51, suggesting that SPE is somehow involved in cell injury.

Classification of CSF samples from normal and post-partum psychotic women using chromatographic profiles with bilinear projections: a multivariate approach.

This paper demonstrates how chromatographic profiles of cerebrospinal fluid (CSF) have been subjected to multivariate data analysis to discriminate between CSF samples from women with post-partum psychosis and those from healthy women. Instead of peak-heights or areas, digitally defined chromatographic profiles were examined using principal component analysis (PCA). In accordance with the diagnosis, we have found a complex profile pattern of at least ten composite peaks that discriminates between these groups. Two of these peaks were for the discrimination particularly clearly between the two groups. We speculate that these findings can be useful in the diagnosis of post-partum psychosis, increasing diagnostic precision and having both clinical and prognostic implications.

Neuropeptide-converting enzymes in cerebrospinal fluid: activities increased in pain from herniated lumbar dis, but not from coxarthrosis.

We measured activities of dynorphin-converting enzyme (DCE), substance P endopeptidase (SPE) and angiotensin-converting enzyme (ACE) in cerebrospinal fluid (CSF) in 13 patients with rhizopathic pain from an herniated lumbar disc, in 9 patients with pain from coxarthrosis and in 11 control patients without pain. In the patients with disc hernia and coxarthrosis, another sample of CSF was analyzed 3-12 months after treatment, when pain had subsided. The DCE activity in the patients was higher than that in both the control patients and the patients with pain from coxarthrosis (nociceptive pain). Similarly, the activity of SPE was lower in the patients with herniated lumbar disc than in controls and in the patients with coxarthrosis. After treatment, the difference in activity compared to controls was lower, but still significant in patients with herniated discs. The ACE activity did not differ from controls in patients with ischialgia, while it was increased in patients with coxarthrosis. This increase also remained after arthroplasty with pain relief. In conclusion, measurements of neuropeptides may be useful for evaluating neuropathic pain.

Isolation of haemorphin-related peptides from filter membranes collected in connection with haemofiltration of human subjects.

This paper describes the extraction and isolation from dialysis filters of two peptides containing the opioid active sequence haemorphin-7. The filter devices were obtained from uraemic patients subjected to haemofiltration. Following acidic extraction of the filter membranes the peptides were purified by size-exclusion, ion-exchange chromatography and finally by reversed-phase chromatography using different columns and different chromatographic systems. The purification was guided by radioimmunoassay and the structure of the final products was elucidated by N-terminal sequencing and fast-atom bombardment mass spectrometry as well as micro-electrospray mass spectrometry. The isolated peptides were suggested to be identical to fragments 1-41 and 32-41 of the beta-chain of human haemoglobin.

Detection of growth hormone receptor mRNA in an ovine choroid plexus epithelium cell line.

The expression of growth hormone receptor (GHR) mRNA in an ovine choroid plexus cell line (SCP) was studied. RNA isolated from SCP cells was subjected to reverse transcription followed by PCR using a set of primers, designed on the basis of the ovine liver GHR sequence. A specific product with expected size of 1204 bp was obtained and the nucleotide sequence was found to be identical to that of ovine liver GHR. When the PCR product was used as a probe for Northern blot analysis, a transcript of 4.4 kb was detected in mRNA isolated from the SCP cells. This is the first report demonstrating the presence of mRNA for GHR in the choroid plexus.

Reversed-phase high-performance liquid chromatography for the determination of haemorphin-like immunoreactivity in human cerebrospinal fluid.

The haemorphins are opioid peptides derived form the blood protein haemoglobin. This study was focused on the detection and determination of haemorphin-like immunoreactivity in human cerebrospinal fluid (CSF) by reversed-phase HPLC. For this purpose a SMART System, optimized for micropurification, was applied. Prior to application to HPLC, the peptide fraction of the CSF sample was extracted using a reversed-phase silica gel cartridge (Sep-Pak C18). In the HPLC separation, the peptide-like material associated with haemorphin-7 immunoreactivity was recovered and determined using a UV detector. The tryptophan residue present in the haemorphin sequence allowed UV detection at wavelengths (e.g., 276 nm) where interference with other co-eluting peptides lacking this residue is minimized. Recorded levels of haemorphin-like immunoreactivity were compared with those detected by radioimmunoassay.

Quantitation and identification of two cholecystokinin peptides, CCK-4 and CCK-8s, in rat brain by HPLC and fast atom bombardment mass spectrometry.

A sensitive HPLC method has been described for quantitation of two cholecystokinin (CCK) peptides in discrete rat brain regions. Separation and quantitation was performed by the reversed-phase HPLC combined with electrochemical detection. Analytical recoveries of the tetrapeptide (CCK-4) and octapeptide-sulphate (CCK-8s) were 96% and 94%, respectively. The between assay coefficient of variation (CV) was less than 3% for both peptides. The within-assay CV was 4% and 6% for CCK-4 and CCK-8s and the detection limit was 2 and 10 pmol/mL, respectively. For identification of structures, the peptides were fractionated by semi-preparative HPLC using a novel SMART system for micropurification. The fractions were analysed by fast atom bombardment mass spectrometry (FAB MS) which confirmed the presence of both CCK-4 and CCK-8s in the rat brain tissue.

Met-enkephalin-Arg6-Phe7 in spinal cord and brain following traumatic injury to the spinal cord: influence of p-chlorophenylalanine. An experimental study in the rat using radioimmunoassay technique.

The possibility that trauma to the dorsal horn may affect the release and distribution of enkephalin was examined using the opioid peptide Met-Enk-Arg6-Phe7 (MEAP) as a marker in a rat model. The peptide content of samples of spinal cord and whole brain was measured using a radioimmunoassay (RIA) technique. In addition, the possible functional relation between this peptide and serotonin was evaluated using a pharmacological approach that included depletion of endogenous serotonin. A focal trauma to the right dorsal horn in the T10-11 segments (2 mm deep and 5 mm long) markedly modified the content of MEAP of the adjacent rostral and caudal segments of the cord, as well as the content of MEAP of the brain. Depletion of serotonin with p-CPA (an inhibitor of the synthesis of serotonin) significantly elevated the content of MEAP in the whole brain without affecting the regions of the spinal cord (except T9 level which showed a 25% decrease from an intact control group). Trauma to the spinal cord in the serotonin-depleted animals did not alter the content of MEAP further, as compared to a p-CPA-treated but untraumatized group. These results indicate that enkephalin (i) participates in the pathophysiology of spinal cord trauma and (ii) suggest that the peptide is somehow functionally related with serotonin.

A comparison of human lung, brain, CSF and plasma angiotensin-converting enzyme with regard to neuropeptide metabolism.

Human ACE obtained from different tissues and body fluids was assayed with regard to degradative action on tachykinins and various opioid peptides. Substance P (1-9) was easily cleaved, whereas substance P and neurokinin A seemed stable against ACE activity. However, endopeptidase-24.11 easily degraded both of these amidated peptides. When the same peptides were assayed as potential inhibitors of the hydrolysis of hippuryl-His-Leu (specific substrate for ACE activity), substance P and its (1-9) fragment were equally potent, whereas neurokinin A was inactive. The beta-casomorphins, beta-casein derived opioid peptides, with a proline residue at their C-terminus also showed inhibitory action on ACE activity, without being cleaved by the enzyme. These results indicate a modulatory action of these peptides. No differences between ACE originating from different tissues or body fluids could be demonstrated in this regard.

Approach to studying proteinase specificity by continuous-flow fast atom bombardment mass spectrometry and high-performance liquid chromatography combined with photodiode-array ultraviolet detection.

Fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography using a photodiode-array ultraviolet detector were applied to study a dynorphin-converting endopeptidase from the human pituitary gland. The specificity of the enzyme was tested towards various opioid peptides derived from the prodynorphin precursor, i.e. dynorphin A, dynorphin B and alpha-neoendorphin. Peptide fragments were analysed directly by continuous-flow FAB-MS and those containing aromatic amino acids were detected independently by the photodiode-array ultraviolet detector. The results obtained suggest a similar processing of these structure-related substrates and it appears that the enzyme recognizes the dibasic stretch in their sequence. It is also clear from this study that the combination of the above techniques provides a powerful tool for studies of enzymatic conversion among the prodynorphin-derived peptides and it should be applicable to studies of similar mechanisms in other peptide systems.

Aminopeptidase in human CSF which degrades delta-sleep inducing peptide (DSIP).

This study describes the purification and characterization of an aminopeptidase from human cerebrospinal fluid capable of degrading delta-sleep-inducing-peptide (DSIP). The enzyme has an apparent molecular weight of approximately 80,000 dalton. It is sensitive towards amastatin, bestatin and EDTA and is optimally active at neutral pH. The recovered enzyme was also found to degrade other neuropeptides, e.g., the enkephalins.