Coexistence of somatostatin receptor subtypes in the human neuroblastoma cell line LA-N-2.

Five distinct human somatostatin receptor subtypes have recently been cloned and characterized. Previous studies have suggested that these receptor subtypes might display coexistent localization, based on in situ hybridization or immunoblockage experiments. Here we provide evidence for coexistence of somatostatin receptor subtypes 2 and 5 in the human neuroblastoma cell line LA-N-2, using a combined approach with RT-PCR and receptor binding studies with somatostatin analogues. Somatostatin receptor subtypes simultaneously localized to a single cell might serve distinct functions in terms of targeting to different intraneuronal compartments or subtype specificity against so far unidentified somatostatin-related peptides.

Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos mRNA in a human neuroblastoma cell line.

Regulation of cholecystokinin (CCK) and the proto-oncogene c-fos mRNA expression was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated either with the tumor promoting phorbol-ester phorbol-12-myristate-13-acetate (PMA), the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c-fos mRNA was determined by Northern-blot analysis with CCK and c-fos specific antisense RNA probes after 4-24 h of drug treatment. Treatment with PMA and IBMX for 4-24 hours transiently raised the CCK mRNA level approximately 1.5-3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK mRNA abundance 1.5-6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c-fos mRNA level approximately 3-5.5 times. The drug combination showed a pronounced synergistic effect and raised the c-fos mRNA level approximately 3-20 times as compared to controls. Apparently, CCK and c-fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP-dependent mechanisms in SK-N-MC cells.

Expression of the cholecystokinin gene in a human (small-cell) lung carcinoma cell-line.

Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence-specific radioimmunoassays in human cell lines. The human small-cell lung carcinoma line (SCLC) U-1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK-N-MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U-1690 cell line. In contrast, the SCLC-line H-69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U-1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U-1690 is a useful model for studies of cell-specific CCK gene expression.

Somatostatinergic phenotype markers in the human neuroblastoma cell-line LA-N-2.

We have characterized somatostatinergic phenotype markers of the human neuroblastoma, LA-N-2. A single mRNA-transcript (approximately 850bp) and two cellular somatostatin immunoreactivity forms, a high molecular weight form (M(r) 15,000) and a fragment corresponding to somatostatin-28 was found, while the somatostatin-14 peptide was absent. Saturation binding experiments demonstrated a single class of high-affinity somatostatin receptors with Kd and Bmax of 0.27 +/- 0.03 nM and 45 +/- 1 fmol/mg protein. Partial G-protein uncoupling (30%) was demonstrated, using GTP gamma S, with an affinity of 9.7 nM. The LA-N-2 cell line, previously shown to be cholinergic, may serve as a simplified system to elucidate heterologous neurotransmittor interactions. Such studies are of interest since dysfunctions of the cholinergic basal forebrain neurons and somatostatin immunoreactive interneurons have been consistently observed in Alzheimer's disease.

cDNA deduced procionin. Structure and expression in protochordates resemble that of procholecystokinin in mammals.

Using an improved 3' RACE (PCR) amplification system containing oligonucleotide primer with an inosine at ambiguous codon positions and inverse PCR to amplify the 5' ends, we have isolated and characterized cDNA clones which encode cionin, a protochordean homologue of the mammalian hormones, cholecystokinin (CCK) and gastrin. The full-length cloned cDNA of 510 bp encoded a 128 amino acid preprocionin. Reverse transcription-PCR and subsequent cDNA cloning revealed that cionin mRNA is expressed in both the neuronal ganglion and the gut of the protochordate Ciona intestinalis. The primary structure of procionin resembles that of proCCK more than that of progastrin. Sequence-specific immunochemical analysis showed that the cionin gene is expressed also at peptide level in both the gut and the neural ganglion. The neuronal processing of procionin is, however, more complete both with respect to carboxyamidation and tyrosine O-sulfation. Hence, the tissue-specific expression of the cionin gene in Ciona intestinalis resembles that of the CCK gene in mammals.

Procholecystokinin and proenkephalin A mRNA expression is modulated by cyclic AMP and noradrenaline.

Regulation of the expression of procholecystokinin (proCCK) and proenkephalin A mRNA was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated with dibutyryl-3',5'-cyclic AMP (dbcAMP), noradrenaline or isoproterenol, a beta-adrenoceptor agonist. Levels of proCCK and proenkephalin A mRNA were determined by Northern blot analysis with proCCK- and proenkephalin A-specific cRNA hybridization probes 9 h after drug treatments. ProCCK and proenkephalin A mRNA were co-expressed in SK-N-MC cells. ProCCK mRNA levels were increased 1.5-2.5 times by dbcAMP, noradrenaline and isoproterenol when compared with controls. The level of proenkephalin A mRNA increased approximately two to three times under the same drug conditions, whereas the level of N-myc mRNA did not change significantly. These results suggest that expression of proCCK and proenkephalin A mRNA may be regulated by a similar cAMP-dependent mechanism in the SK-N-MC cell line.

Acupuncture mechanisms in rabbits studied with microinjection of antibodies against beta-endorphin, enkephalin and substance P.

Injection of protein-A purified antibodies against Met-enkephalin and beta-endorphin into the periaqueductal gray matter (PAG) was shown to decrease the analgesic effect of electroacupuncture (EA) in rabbits. Met-enkephalin antibodies were more potent than the beta-endorphin antibodies in causing a statistically-significant effect on electroacupuncture analgesia. Antibodies to Met-enkephalin were also active at the spinal level, whereas antibodies against beta-endorphin were without effect: this is in agreement with a rich enkephalinergic innervation and absence of beta-endorphin-containing fibres in the spinal cord. Substance P, the other neuropeptide of this study, also seems to be important in mediating effects of electroacupuncture. Injection of antibodies into the periaqueductal gray caused decrease of the effect of electroacupuncture whereas intrathecal administration of Fab-fragment substance P antibodies caused a marked potentiation. The demonstration of site specificity of the neuropeptides in mediating analgesia induced by electroacupuncture supports the validity of this experimental approach.

Modulation of proenkephalin A gene expression by cyclic AMP.

Regulation of proenkephalin A expression was studied in the human neuroblastoma SK-N-MC cell line with respect to mRNA-level, translation, posttranslational processing of the prohormone and secretion of the processed products into the culture medium. Cells were treated with either norepinephrine (NE), dexamethasone (DEX), dibutyryl-3',5'-cyclic AMP (dbcAMP) or the combination of NE and DEX. In an additional investigation, proenkephalin A mRNA levels were determined after 9 h of treatment with dbcAMP, NE, isoproterenol, NE + propranolol and dbcAMP + DEX. NE or dbcAMP for 1-48 h transiently elevated proenkephalin A mRNA 1.5-4.5 times compared to control. The effect of NE was partially blocked by the beta-adrenoceptor antagonist propranolol and was reproduced by the beta-adrenoceptor agonist isoproterenol, suggesting involvement of the beta-adrenoceptor. DEX alone had no significant effect. However it markedly antagonized the effect of NE but not that of dbcAMP suggesting an action on the beta-adrenoceptor. The intracellular content of Met-enkephalin-Arg6,Phe7 immunoreactivity was increased during drug treatment in parallel with changes in proenkephalin A mRNA. DEX gave no effect. No significant change in the ratio of low versus high molecular weight immunoreactive material could be detected in the cell extracts as determined at different time points. Secretion of immunoreactivity into the culture medium increased 5-fold after 18 h of treatment with NE, whereas dbcAMP gave a 2-fold increase. The proportion of low-molecular weight secreted material increased markedly. DEX alone did not induce any change but inhibited the effect of NE. Apparently, regulation of gene expression, prohormone processing and secretion are coordinated by a cAMP-dependent mechanism.

Enzyme-linked immunosorbent assay of substance P and its metabolite SP 1-7. A comparison with RIA.

We have investigated enzyme-linked immunosorbent assay (ELISA) as a possible alternative to radioimmunoassay (RIA) for the detection and measurement of the neuropeptide substance P (SP) and its metabolite substance P1-7. The sensitivities were higher with ELISA than with RIA utilizing the same antisera. The intra-assay and inter-assay variation in ELISA was 1% and 13% respectively. The higher sensitivities are in part due to the standard curves having less steep slopes, and in part to lower IC50s in the ELISA. Since there was a good correlation between peptide levels in biological samples as determined by ELISA and RIA respectively, ELISA might be considered an attractive alternative to RIA.

Alzheimer's disease: molecular genetics and transgenic animal models.

Disease-causing mutations in the amyloid precursor protein (APP) gene have been found on chromosome 21 during the last 2 years in some early onset Alzheimer's disease (AD) families. Genetic evidence shows that other genes than the APP are also involved in the aetiology of AD. Linkage to a loci on chromosome 14 has been found in early onset disease. The identification of APP mutation has led to the realization that APP mismetabolism is a central event in the aetiology and pathogenesis of the disease. Experiments to test this in transgenic mice have so far met with little success. There are many possible explanations for the problems to generate transgenic mice. These include the possibilities that mice are incapable of developing AD for reasons dependent on their APP sequence; and that appropriate regulation of APP gene is required for pathology to develop. Current attempts that seem promising to model the disease pathology are the use of homologous recombination to insert the pathogenic mutation and transfection of YACs into transgenic animals.

Expression of the proenkephalin gene in human neuroblastoma cell lines.

Several human tumour cell lines were screened for secretion of proenkephalin-derived peptides with an antiserum directed to its N-terminus, Met-enkephalin-Arg6,Phe7 and for proopiomelanocortin-derived peptides with an antiserum to beta-endorphin. The neuroblastoma SK-N-MC cell line secreted Met-enkephalin-Arg6,Phe7-immunoreactive peptides in relatively high amounts into the culture medium, although processing was not complete and there was no evidence for free Met-enkephalin-Arg6,Phe7. Gene expression was confirmed by the presence of proenkephalin mRNA and proenkephalin-derived polypeptides in extracts of the SK-N-MC cells and also in the neuroblastoma SH-SY5Y cell line. In the latter cells, however, the expression was approximately 3 times lower, there was less processing of proenkephalin and no evidence for secretion.

Environmental influence on somatostatin levels and gene expression in the rat brain.

In the present study we have quantified preprosomatostatin-mRNA and somatostatin levels in rat brain following environmental stimulation. Animals were housed for 30 days in an enriched or impoverished environment prior to analysis. After 30 days of housing half of the rats from each environment were behaviourally tested for 3 days. Housing in enriched environment improved performance in a spatial learning situation. The open-field behaviour of these animals was characterized by initially higher rearing scores and a more rapid habituation to novel environment as measured by spontaneous locomotor activity. We found significantly elevated somatostatin levels in the cortex following enriched environment, compared with impoverished environment. Exposure to behavioural testing of impoverished animals led to increased cortical somatostatin levels. Hypothalamic somatostatin levels increased significantly after housing in enriched environment, while the testing procedure had no influence. Our data shows that the somatostatin system in the rat brain was activated in association with cognitive changes, that were induced by housing in an enriched environment.

Enkephalins interact with substance P-induced aversive behaviour in mice.

The effect of the endogenous opioid peptides, methionine-enkephalin (Met-ENK), beta-endorphin (beta-END) and dynorphin-(1-17) (DYN) on the aversive behavior produced by intrathecal (i.t.) administration of substance P (SP) was studied in mice. A low dose of i.t. administered Met-ENK gave a marked reduction of the SP-induced response. In the tail-flick assay, such doses of Met-ENK were ineffective in producing antinociception. At much higher doses, however, Met-ENK obtained antinociceptive activity. In contrast, beta-END and DYN had about the same potency in inhibiting the SP-induced behavioural response and in the tail-flick test, respectively. These results suggest that opioid peptides, particularly enkephalin neurons in the spinal cord influence SP-induced aversive behaviour.

Proenkephalin A-like mRNA in human leukemia leukocytes and CNS-tissues.

Total RNA has been prepared from human leukocytes from patients with chronic lymphoblastic leukemia (CLL) as well as from post mortem human caudate nucleus, hypothalamus, cerebellum and cerebral cortex. Dot-blot and Northern blot analysis, using a human proenkephalin A clone and SP-6 derived "complementary" proenkephalin A RNA respectively, revealed the existence of proenkephalin A-like RNA:s in CLL-leukocytes with the same characteristics as in caudate nucleus, hypothalamus, and cortex. Furthermore, RIA and Western blot analysis confirmed that immunoreactive pro-enkephalin A activity is present in human CLL-leukocytes. The progress in DNA recombinant technology has allowed the study of opioid peptide regulation at the transcriptional and translational-posttranslational level. Studies on the distribution and quantitation of preproenkephalin A mRNA in bovine, rat and human central nervous system (CNS) have recently been reported. Different opioid peptides, related to the enkephalins, dynorphins and beta-endorphin have also been detected in tissues outside the CNS including the adrenal medulla and in pheochromocytomas. Northern blot analysis and cDNA-cloning confirmed that the proenkephalin A gene is indeed expressed in these tissues. Proenkephalin A derived peptides are potentially significant in nervous disorders. We have chosen to investigate whether the corresponding gene is expressed not only in CNS-tissues but also in human leukocytes, cells readily obtained in individual patients.

An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P.

The measurements of peptides of the enkephalin, dynorphin and substance P systems is complicated by the number of possible precursor fragments and degradation products that might cross-react with the antisera. By using an ion-exchanger step before radioimmunoassay we can reduce the possibility that observed peptide levels are due to precursors or metabolites. The ion-exchanger method runs with good recovery and its main advantage is that many samples can be run in parallel. The recovery from the ion-exchanger was similar using two different homogenizing media, whereas the measured endogenous levels of [Met] and [Leu]enkephalin were 3-4 fold higher with 1M acetic acid than when a 1:1 MeOH/HCl mixture was used for tissue extraction.

Downregulation of uncoupling protein 2 mRNA in women treated with glucocorticoids.

OBJECTIVE: Glucocorticoids are well-known regulators of energy turnover and adipose tissue metabolism. We investigated the effect of glucocorticoids on the expression of the human uncoupling protein 2 (UCP 2) gene, which has been implicated in energy expenditure. DESIGN: Prednisolone (25 mg) was administered orally daily for 7 days. Subcutaneous adipose tissue UCP 2 mRNA was measured before and after treatment. SUBJECTS: Eight healthy female subjects (age 52-63 y; body mass index 25-34 kg/m2). RESULTS: No differences in body weight, waist-to-hip ratio or plasma-values of FFA or glucose were found after prednisolone treatment, as compared to pre-treatment values under these conditions. In contrast, plasma insulin levels were significantly increased by glucocorticoid administration, 54+/-6 before vs 70+/-12 (mean+/-sem) pmol/l after treatment (P=0.028). Furthermore, using RT-competitive-PCR, the UCP 2 mRNA level in abdominal subcutaneous adipose tissue was found to be down-regulated by half (6.3+/-0.4 vs 3.1+/-0.8 amol/microg RNA, P=0.012) after glucocorticoid treatment. No difference in expression levels of the reference gene 18SrRNA was observed before, as compared to after prednisolone exposure (249+/-11 vs 248+/-30 amol/microg RNA, P=0.87). CONCLUSION: These data suggest that glucocorticoids may play a role in the regulation of UCP 2 mRNA expression in human adipose tissue in vivo.

[Met]enkephalin-Arg6-Phe7 in human CSE--increased levels in late pregnancy.

The opioid heptapeptide, [Met]enkephalin-Arg6-Phe7 was measured in CSF of women at term pregnancy and during the luteal phase by RIA. Levels turned out to be significantly increased in pregnant women. These results are in agreement with an earlier paper in which we reported increased levels of receptorassayed opioid peptides deriving from proenkephalin A in CSF of women in term pregnancy compared to levels in nonpregnant, nonpuerperal women.

A marked upregulation of uncoupling protein 2 gene expression in adipose tissue of hyperthyroid subjects.

Recently, a family of uncoupling protein (UCP) genes has been discovered. The role of these genes is unknown, but it has been suggested that they are involved in regulating resting metabolic rate. In this study, we hypothesised that thyroid hormone status may influence the expression of UCP2 mRNA. The adipose tissue levels of UCP2 mRNA were measured in eight female subjects before and after treatment for thyrotoxicosis. All subjects in the hyperthyroid condition had markedly enhanced plasma levels of thyroxine (62.0 +/- 6.9 vs. 17.9 +/- 1.7, p = 0.012) and triiodothyronine (37.9 +/- 6.9 vs. 5.9 +/- 0.9, p = 0.012), accelerated heart rate (94 +/- 7 vs. 69 +/- 5, p = 0.012), decreased BMI (24.5 +/- 1.9 vs. 25.1 +/- 1.9, p = 0.025) and decreased percentage body fat (32.8 +/- 4.4 vs. 37.1 +/- 4.5, p = 0.018), as compared to the euthyroid state. Using RT-competitive-PCR, the UCP2 mRNA levels were found to be 2.5-fold upregulated in hyperthyroidism (10.4 +/- 1.7 vs. 4.2 +/- 1.3 amol/microg RNA, p = 0.012). In contrast, no difference in expression levels of the reference gene 18SrRNA was seen in the hyperthyroid versus the euthyroid state (317 +/- 49 vs. 279 +/- 25 amol/microg RNA, p = 0.48) but the difference in UCP2 mRNA levels between the hyper- and euthyroid state remained when UCP2 was related to 18SrRNA (p = 0.012). In conclusion, thyrotoxicosis markedly increases the expression of UCP2 mRNA in adipose tissue, which suggests a role for thyroid hormones in the regulation of this uncoupling protein in man.

Cerebrospinal fluid concentrations of substance P and (met)enkephalin-Arg6-Phe7 during surgery and patient-controlled analgesia.

The possible role of two neuropeptides (substance P and (Met)enkephalin-Arg6-Phe7) in nociception were studied in 14 surgical patients. Lumbar cerebrospinal fluid (CSF) concentrations of the putative excitatory afferent transmitter substance P and the mu and delta receptor agonist (Met)enkephalin-Arg6-Phe7 were measured during general anesthesia for abdominal surgery and during the postoperative period when patient-controlled analgesia (PCA) was used for control of pain. The CSF was sampled through an intrathecal catheter. Seven of the patients were randomly assigned to receive neurolept anesthesia; the rest were given isoflurane anesthesia without narcotics. No statistically significant changes occurred in substance P concentrations in CSF during surgery or postoperative PCA, nor were there significant differences between the two groups. There was, however, a significant correlation between CSF substance P concentrations before the start of PCA and pain assessment on a visual analogue scale. The individual changes in substance P concentrations during PCA was also inversely correlated to the consumption of meperidine. The CSF (Met)enkephalin-Arg6-Phe7 concentrations were below the level of detection in seven of the patients before anesthesia. A large interindividual variability in both substance P and (Met)enkephalin-Arg6-Phe7 concentrations was evident. The absence of major changes in CSF neuropeptide concentrations was unexpected. Apparently inter-individual variations in neuropeptide output are considerable.