Menopausal hormone therapy and biliary tract cancer: a population-based matched cohort study in Sweden.

BACKGROUND: This study tested the hypothesis that contemporary menopausal hormonal therapy (MHT) increases the risk of biliary tract cancer. The risk of cancer of the biliary tract (gallbladder and extra-hepatic bile ducts) may be increased following estrogen exposure. MATERIAL AND METHODS: This was a nationwide population-based matched cohort study in Sweden. Data from the Swedish Prescribed Drug Register identified all women exposed to systemic MHT in 2005-2012. Group-level matching (1:3 ratio) was used to select women unexposed to MHT from the same study base, matched for history of delivery, thrombotic events, hysterectomy, age, smoking- and alcohol related diseases, obesity, and diabetes. Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Comparing 290,186 women exposed to MHT with 870,165 unexposed, MHT did not increase the OR of biliary tract cancer. The OR of gallbladder cancer was rather decreased in MHT users (OR 0.58, 95% CI 0.43-0.79), but this association became attenuated and statistically non-significant after adjusting for gallstone disease (OR 0.84, 95% CI 0.60-1.15). The OR of extra-hepatic bile duct cancers was 0.83 (95% CI 0.61-1.15). There were no clear differences when the analyses were stratified for estrogen or estrogen/progestogen combinations. MHT increased the risk of gallstone disease (OR 6.95, 95% CI 6.64-7.28). CONCLUSIONS: Contemporary MHT does not seem to increase the risk of biliary tract cancer. The decreased risk of gallbladder cancer may be explained by the increased use of surgery for symptomatic gallstones in MHT users.

Different menopausal hormone regimens and risk of breast cancer.

Background: There are considerable knowledge gaps concerning different estrogen and progestin formulations, regimens, and modes of administration of menopausal hormone therapy (HT) and the risk of breast cancer. Our objective was to assess the different treatment options for menopausal HT and the risk of breast cancer. Patients and methods: This Swedish prospective nationwide cohort study included all women who received ≥1 HT prescription during the study period 2005-2012 (290 186 ever-users), group-level matched (1 : 3) to 870 165 never-users; respectively, 6376 (2.2%) and 18 754 (2.2%) developed breast cancer. HT, ascertained from the Swedish Prescribed Drug Register, was subdivided by estrogen and progestogen formulation types, regimens (continuous versus sequential) and modes of administration (oral versus transdermal). The risk of invasive breast cancer was presented as adjusted odds ratios (OR) and 95% confidence intervals. Results: Current use of estrogen-only therapy was associated with a slight excess breast cancer risk [odds ratio (OR) = 1.08 (1.02-1.14)]. The risk for current estrogen plus progestogen therapy was higher [OR = 1.77 (1.69-1.85)] and increased with higher age at initiation [OR = 3.59 (3.30-3.91) in women 70+ years]. In contrast, past use was associated with reduced breast cancer risk. Current continuous estrogen/progestin use was associated with higher risk [OR = 2.18 (1.99-2.40) for progesterone-derived; OR = 2.66 (2.49-2.84) for testosterone-derived] than sequential use [OR = 1.37 (0.97-1.92) for progesterone-derived; OR = 1.12 (0.96-1.30) for testosterone-derived]. The OR for current use was 1.12 (1.04-1.20) for estradiol, 0.76 (0.69-0.84) for estriol, 4.47 (2.67-7.48) for conjugated estrogens, and 1.68 (1.51-1.87) for tibolone. Oral and cutaneous HT showed similar associations. Conclusion: Different HT regimens have profoundly different effects on breast cancer risk. Because of registry limitations some confounders could not be assessed. This knowledge may guide clinical decision-making when HT is considered.

Mortality from laparoscopic antireflux surgery in a nationwide cohort of the working-age population.

BACKGROUND: Both medication and surgery are effective treatments for severe gastro-oesophageal reflux disease (GORD). Postoperative risks have contributed to decreased use of antireflux surgery. The aim of this study was to assess short-term mortality following primary laparoscopic fundoplication. METHODS: This was a population-based nationwide cohort study including all Swedish hospitals that performed laparoscopic fundoplication between 1997 and 2013. All patients aged 18-65 years with GORD who underwent primary laparoscopic fundoplication during the study interval were included. The primary outcome was absolute all-cause and surgery-related 90- and 30-day mortality. Secondary outcomes were reoperation and length of hospital stay. Logistic regression was used to calculate odds ratios with 95 per cent confidence intervals of reoperation within 90 days and prolonged hospital stay (4 days or more). RESULTS: Of 8947 included patients, 5306 (59·3 per cent) were men and 551 (6·2 per cent) had significant co-morbidity (Charlson score above 0). Median age at surgery was 48 years, and median hospital stay was 2 days. The annual rate of laparoscopic fundoplication decreased from 15·3 to 2·4 patients per 100 000 population during the study period, whereas the proportion of patients with co-morbidity increased more than twofold. All-cause 90- and 30-day mortality rates were 0·08 per cent (7 patients) and 0·03 per cent (3 patients) respectively. Only one death (0·01 per cent) was directly surgery-related. The 90-day reoperation rate was 0·4 per cent (39 patients). Co-morbidity and older age were associated with an increased risk of prolonged hospital stay, but not reoperation. CONCLUSION: This population-based study revealed very low mortality and reoperation rates following primary laparoscopic fundoplication in the working-age population. The findings may influence clinical decision-making in the treatment of severe GORD.

Art meets science: The Cosmopolitan Chicken Research Project.

The Cosmopolitan Chicken Project is an artistic undertaking of renowned artist Koen Vanmechelen. In this project, the artist interbreeds domestic chickens from different countries aiming at the creation of a true Cosmopolitan Chicken as a symbol for global diversity. The unifying theme is the chicken and the egg, symbols that link scientific, political, philosophical and ethical issues. The Cosmopolitan Chicken Research Project is the scientific component of this artwork. Based on state of the art genomic techniques, the project studies the effect of the crossing of chickens on the genetic diversity. Also, this research is potentially applicable to the human population. The setup of the CC®P is quite different from traditional breeding experiments: starting from the crossbreed of two purebred chickens (Mechelse Koekoek x Poule de Bresse), every generation is crossed with a few animals from another breed. For 26 of these purebred and crossbred populations, genetic diversity was measured (1) under the assumption that populations were sufficiently large to maintain all informative SNP within a generation and (2) under the circumstances of the CCP breeding experiment. Under the first assumption, a steady increase in genetic diversity was witnessed over the consecutive generations, thus indeed indicating the creation of a "Cosmopolitan Chicken Genome". However, under the conditions of the CCP, which reflects the reality within the human population, diversity is seen to fluctuate within given boundaries instead of steadily increasing. A reflection on this might be that this is because, in humans, an evolutionary optimum in genetic diversity is reached. Key words.

Laryngeal abductor reinnervation with a phrenic nerve transfer after a 9-month delay.

BACKGROUND: Successful restoration of laryngeal abductor function, using the phrenic nerve, has been described in the cat model in the acute phase. However, in clinical practice there is usually a considerable delay between injury to the RLN and presentation for treatment. Delayed reinnervation therefore would be more suitable in clinical practice. OBJECTIVE: To test the feasibility of delayed selective abductor reinnervation following transection of the recurrent laryngeal nerve (RLN). MATERIALS AND METHODS: In 12 cats, the right RLN was severed. Nine months later, the phrenic nerve was anastomosed to the distal RLN stump with all its branches directed toward the posterior cricoarytenoid muscle. For 10 weeks after the reconstruction, electromyography and videolaryngoscopy were performed weekly. Finally, histological analysis of the RLN was performed. RESULTS: Evaluation was possible in 11 cats. Reinnervation of the right posterior cricoarytenoid muscle with the phrenic nerve occurred in 10 cats following nerve anastomosis, but results of videolaryngoscopy showed adequate to good abduction in only 4 cats. The main limiting factor was reduced mobility of the cricoarytenoid joint. Evidence of spontaneous subclinical reinnervation after the delay was observed in 7 cats but apparently did not impede the surgical reinnervation. CONCLUSIONS: Delayed selective laryngeal abductor reinnervation was feasible, but function recovery was less successful than if performed immediately. Future investigations should concentrate on early determinants of spontaneous restoration of function to allow early selection of patients who are eligible for reinnervation surgery.

Selective laryngeal reinnervation with separate phrenic and ansa cervicalis nerve transfers.

OBJECTIVE: To perform selective reinnervation of the laryngeal abductor and adductor muscle groups after injury to the recurrent laryngeal nerve, recovering laryngeal function without impairment by synkinesis. DESIGN: Ten cats underwent the surgical procedure. To reinnervate the posterior cricoarytenoid muscle (abductor), a phrenic nerve graft was anastomosed to the main trunk of the recurrent laryngeal nerve. The adductor branch was severed, and the proximal stump was buried in the posterior cricoarytenoid muscle. The sternohyoid branch of the ansa cervicalis was anastomosed to the distal stump to reinnervate the adductor muscle group. After a period of 10 weeks, the laryngeal function was evaluated with videolaryngoscopy and electromyography of the posterior circoarytenoid and vocalis muscles. RESULTS: Of the 10 cats, 9 could be evaluated. Laryngeal abductor function was comparable with the unaffected side in the 9 cats. During respiratory distress conditions, a minor compromise of the maximal abduction was observed in 5 cats. Phonation was not tested, but spontaneous adduction during expiration was seen in all cats. Reflex closure on ipsilateral, supraglottic, tactile mucosal stimulation was seen in only 2 cats. In each cat, evidence of nerve regeneration and reinnervation of both muscle groups was established with electromyography, electrical stimulation, and histological examination. CONCLUSIONS: Using this selective reinnervation procedure, good laryngeal function can be achieved in the cat model, which may be applicable in humans. By reinnervation of the vocalis muscle, muscle tonus is achieved, which is expected to improve voice quality. Using this procedure, however, no active reflex closure may be expected.

The use of macroporous microcarriers for the large-scale growth of V79 cells genetically designed to express single human cytochrome P450 isoenzymes and for the characterization of the expressed cytochrome P450.

In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 x 10(5) cells/ml, approximately 1 x 10(7) cells/ml could easily be harvested after 6 days in the case of the V79h1A1 cells, resulting in a total of 3.6 x 10(10) cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 +/- 7.9 vs 14.5 +/- 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6 beta-hydroxylation activities. EROD, ECOD, and testosterone-6 beta-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6 beta-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.

Laryngeal abductor function after recurrent laryngeal nerve injury in cats.

OBJECTIVE: To determine the influence of severity of neural injury of t he recurrent laryngeal nerve on recovery of laryngeal abductor function and the importance of synkinesis. DESIGN: The recovery of laryngeal abductor function was studied in 30 cats after crushing (second-degree injury) or transection followed by neurorrhaphy (fifth-degree injury) of the recurrent laryngeal nerve, with a reinnervation period of 10 weeks. MAIN OUTCOME MEASURES: Recovery of laryngeal abductor function was evaluated by videolaryngoscopy of spontaneous laryngeal abduction during respiration and electromyography of the posterior cricoarytenoid and vocalis muscles. Neural lesions were applied unilaterally, and recovery of laryngeal function was compared with the contralateral unimpaired hemilarynx. Reinnervation was confirmed by histologic examination. RESULTS: After the recurrent laryngeal nerve was crushed, laryngeal abductor function was similar to normal after a 10-week reinnervation period in 19 of the 20 cats; after neurorrhaphy, no notable recovery of laryngeal abduction resulted in any of 10 cats. Electromyographic recordings disclosed synkinesis after neurorrhaphy and recovery of normal activity patterns after crush injuries. CONCLUSIONS: Severity of neural injury to the recurrent laryngeal nerve influences the recovery of laryngeal abductor function. Damage to the endoneurium leads to misdirection of regenerating axons, inappropriate reinnervation, and synkinesis. No effective laryngeal function can then be expected.

Typical and atypical fractures of the odontoid process in young children. Report of two cases and a review of the literature.

The most common injury to the odontoid process in children under the age of seven years is a fracture through the synchondrosis with or without anterior displacement of the odontoid process, but this is not the only type of fracture of the odontoid process in this age-group. Fractures above and below the synchondrosis and fractures with posterior displacement were described. Typical clinical features of these fractures are: (1) major and blunt trauma, (2) neck pain and resistance to active and passive head movements; and (3) no or only slight neurological deficits. Conservative treatment had excellent results in the majority of cases. Nevertheless, there are a few specific indications for surgery.

SR 57746A attenuates cytostatic drug-induced reduction of neurite outgrowth in co-cultures of rat dorsal root ganglia and Schwann cells.

A co-culture system of intact rat dorsal root ganglia (DRG) with Schwann cells was used to evaluate the potential neurotrophic activity of SR 57746A. Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of SR 57746A. Neurite outgrowth of intact DRG was increased by SR 57746A and this was more obvious in the presence of co-cultured Schwann cells. The neuroprotective properties of SR 57746A were studied in co-cultures of DRG and Schwann cells, in which neuritogenesis was reduced by the cytostatic drugs cisplatin, vincristine and taxol. It was found that neurite outgrowth from DRG treated with cisplatin (3 micrograms/ml) and 10 microM SR 57746A for 3 days was significantly higher than after treatment with cisplatin alone. Similarly, neuritogenesis from DRG treated with taxol (0.01 microgram/ml) or vincristine (0.5 ng/ml) in combination with 10 microM SR 57746A was significantly increased compared to treatment with taxol or vincristine alone. When intact DRG were incubated in vitro with 3 micrograms/ml cisplatin and without Schwann cells, 10 microM SR 57746A also had a neuroprotective effect. These data suggest that SR 57746A has neuroprotective potential and that this effect does not depend solely on the presence of Schwann cells.

Combined in situ hybridisation, northern blot analysis, and receptor binding studies in clones expressing different levels of the human 5-HT1A receptor.

In order to set up the technique of semi-quantitative in situ hybridisation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expressing the human 5-HT1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of stably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 showed saturable binding for the agonist [3H]-8-OH-DPAT, with receptor densities (Bmax) of 227 +/- 86, 548 +/- 107 and 1505 +/- 212 fmol/mg protein respectively, and with corresponding affinity constants (pKd) of 8.8 +/- 0.1, 9.1 +/- 0.1, and 9.1 +/- 0.1 nM, respectively. Northern blot analysis using a specific probe for the 5-HT1A receptor revealed the presence of a single 1.56 kilobase mRNA species in the 5-HT1A receptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT1A receptor mRNA levels in these three 5-HT1A transfectants using [35S]alphaCTP labeled riboprobes (sense and anti-sense). The following rank order of receptor mRNA expression was found for clones 66, 53 and 47 respectively: 0.140 +/- 0.001, 0.365 +/- 0.045 and 0.835 +/- 0.115 (relative optical density units). With the sense probe no specific labelling was observed. In conclusion, a positive correlation was found between receptor density (Bmax) and receptor mRNA expression (semi-quantitative in situ hybridisation) using human 5-HT1A receptor clones with different expression levels.

Morphometric analysis of cisplatin-induced neurite outgrowth in N1E-115 neuroblastoma cells.

Cisplatin, a widely used cytostatic drug for the control of a variety of neoplastic tumors, unexpectedly induced neurite outgrowth in N1E-115 neuroblastoma cells and this phenomenon was studied further in detail with morphometric analysis. As expected, cisplatin dose-dependently reduced cell number. At the same time, however, cisplatin affected the morphology of the neuroblastoma cells that changed from small rounded cell bodies into large flat cell bodies with neurites. The neurite length/cell as a function of cisplatin concentration showed a bell-shaped curve. The maximal effect (1200% of control) on neurite length/cell was observed at 1 microgram/ml cisplatin. In conclusion, cisplatin induced cellular differentiation in N1E-115 neuroblastoma cells at and just above threshold doses for cytostatic activity.

Alpha-sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of laminin.

Schwann cells play an important role in peripheral nerve regeneration. Here, we report the effect of alpha-sialyl cholesterol (alpha-SC), a derivative of the sialic acid-containing natural gangliosides, and the cytostatic agents, cisplatin, taxol and vincristine on the laminin production in Schwann cell cultures isolated from rat sciatic nerves. Laminin, one of several extracellular matrix components produced by Schwann cells, is known to potentiate axonal outgrowth. Laminin content was increased by alpha-SC, starting at 7.0 micrograms/ml with a maximal effect at 22.4 micrograms/ml (30%, P < 0.001). The three cytostatic drugs, dose-dependently reduced laminin content in Schwann cell cultures: (1) cisplatin at a threshold dose of 2 micrograms/ml (-26.4%, P < 0.001); (2) taxol, starting at a dose of 1 ng/ml (-8.0%, P < 0.05); and (3) vincristine, starting at 0.5 ng/ml (-5.9%, P < 0.05). Cultured Schwann cells were incubated with cytostatic drugs in combination with increasing amounts of alpha-SC and it was found that, depending on the cytostatic drug concentration used, alpha-SC could reduce or completely prevent the cytostatic drug-induced reduction of laminin in Schwann cell cultures. Co-treatment with alpha-SC also reduced part of the morphological changes caused by the cytostatic drugs. alpha-SC did not counteract the anti-proliferative effect of the cytostatic drugs on K-562 human erythroleukemia cells. In conclusion, alpha-SC increased laminin content in Schwann cell cultures and protected Schwann cell cultures against the decrease of laminin by cytostatic drugs without interfering with the anti-proliferative potential, suggesting that alpha-SC may have clinical use in protecting cancer patients against the neurotoxic effects of cytostatic drugs.

Reversal by NGF of cytostatic drug-induced reduction of neurite outgrowth in rat dorsal root ganglia in vitro.

Cytostatic drugs, like cisplatin, vincristine and taxol, when given to cancer patients may cause peripheral neuropathies. We were interested in the potential neuroprotective effects of neurotrophic factors against such neuropathies. To this aim we studied the effects of these cytostatic agents on sensory fibers located in the dorsal root ganglia (DRG) in vitro and studied whether nerve growth factor (NGF) could reverse the cytostatic induced morphological changes on intact DRG (1 DRG/well, n = 10 per dose). Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of cytostatic drugs in the presence of 3 ng NGF/ml and cytosine arabinoside (Ara-C, 10(-6) M). Relative neurite outgrowth in intact DRG in culture was reduced dose-dependently, (a) by vincristine starting at a dose of 0.4 ng/ml for 2 days (-33% as compared to control; P < 0.001, Student's t-test); (b) by taxol 10 ng/ml (-60%; P < 0.001), and (c) by cisplatin 3 micrograms/ml (-47%, P < 0.001). Cisplatin also prevented the migration of satellite cells away from the intact DRG along the extending neurites into the well in contrast to control, vincristine, or taxol. To evaluate the neuroprotective potential of NGF in this in vitro cytostatic neuropathy model, we incubated intact DRG with cytostatic agents in combination with increasing amounts of NGF. Neurite outgrowth from DRG treated with vincristine (0.5 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+87%) than after treatment with vincristine + 1 ng NGF/ml (P < 0.001). Neurite outgrowth from DRG treated with taxol (20 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+228%) than after taxol + 1 ng NGF/ml (P < 0.05). Neuritogenesis from DRG treated with cisplatin (2.5 micrograms/ml)+NGF (3 ng/ml) for 2 days was significantly increased (+105%) compared to treatment with cisplatin + 1 ng NGF/ml (P < 0.001). DRG thus appear to be a very suitable model for studying cytostatic drug-induced neuropathies in vitro and NGF has a clear neuroprotective effect on the vincristine-, taxol-, and cisplatin-induced neuropathies in this in vitro model.

Regulation of CCK mRNA in the human neuroepithelioma cell line SK-N-MCIXC in response to second messenger activators.

Regulation of cholecystokinin (CCK) expression was studied in the human neuroepithelioma cell line SK-N-MCIXC. The cells were treated with the phosphodiesterase inhibitor isobutyl-methylxanthine and the tumor promoting phorbol ester, phorbol-12-myristate 13-acetate; activators of the cyclic AMP (cAMP) and protein kinase C (PKC) second messenger pathways, respectively. Levels of CCK mRNA were determined after 6, 12 and 24 hour drug treatments, with Northern blot analysis using human CCK cDNA hybridization probes. Activation of both cAMP and PKC second messenger pathways increased CCK mRNA levels in SK-N-MCIXC cells. These results indicate that the levels of CCK mRNA in SK-N-MCIXC cells are regulated by cAMP and PKC dependent mechanisms.

Processing, release and metabolism of cholecystokinin in SK-N-MCIXC cells.

The human cholinergic neuroepithelioma cell line SK-N-MCIXC, which expresses high levels of cholecystokinin (CCK) mRNA and secretes intact CCK into the media, was used to examine CCK processing and metabolism. Our data provide evidence for the existence of specific candidate processing enzymes in SK-N-MCIXC cells which may be involved in processing proCCK in the brain and indicate that SK-N-MCIXC cells provide a model system for studying the regulation of these enzymes. mRNAs for the intracellular processing enzymes, prohormone convertase 1 (PC1), PC2 and furin were present in SK-N-MCIXC cells. PC1 and/or PC2 and/or furin may cleave at the dibasic amino acid pairs Arg-Arg at the C-terminal part of proCCK, and Arg-X-X-Arg at the N-terminal of the CCK-58 sequence in proCCK. The SK-N-MCIXC cell line demonstrated spontaneous and regulated release of CCK and large amounts of CCK-precursors, as measured with region specific radioimmunoassays coupled to high performance liquid chromatography. Storage granules containing glycine-extended CCK were shown in SK-N-MCIXC cells using indirect immunofluorescence. The extracellularly localized CCK-metabolizing enzyme, neutral endopeptidase 24.11 (EC 3.4.24.11), was present in membranes from both SK-N-MCIXC cells and in intact slices of rat cerebral cortex. The rat cerebral cortex is a brain region known to be rich in CCK. The SK-N-MCIXC cell line provides an in vitro model to study the regulation of CCK synthesis and metabolism in neuronal systems since it contains the storage granules, mRNA, intact peptide, and complement of enzymes necessary for biosynthesis and metabolism of CCK.

Peptidases in the CNS: formation of biologically active, receptor-specific peptide fragments.

Peptides function as chemical signals between cells of multicellular organisms, or different organisms, via specific receptors on target cells. Many hormones, neuromodulators, and growth factors are peptides. Because there is no known reuptake system for peptides at the nerve terminal, the biological activity of peptides in the extracellular space is regulated by enzymatic degradation and extracellular metabolism. For example, angiotensin I is processed extracellularly in the lung by angiotensin-converting enzyme (ACE; E.C. 3.4.15.1), a peptidyl dipeptidase, to form the potent vasoconstrictor hormone angiotensin II. When neuropeptides are released from neurons into the extracellular space, specific peptidases also can modulate the peptidergic signal by generating smaller, biologically active fragments via products with similar or dissimilar characteristics of the parent peptide. Therefore, receptor-binding selectivity of a released peptide hormone can be regulated by peptidases. Because peptidases may play a key role in the extracellular regulation of peptidergic signaling, alterations in peptidase activities by drugs or disease states may lead to disruptions in biological homeostasis. The subject of this article is the role of peptidases in the central nervous system in the formation of biologically active, receptor-specific peptides from peptide E, beta-endorphin, neurotensin, and cholecystokinin.

Specificity of neurotensin metabolism by regional rat brain slices.

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.

A specific enzyme assay for aminopeptidase M in rat brain.

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 microM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 microM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 microM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.

Chronic haloperidol and chlorpromazine treatment alters in vitro beta-endorphin metabolism in rat brain.

To determine if chronic haloperidol (3.0 mg/kg per day) or chlorpromazine (4.2 mg/kg per day) treatment alters central beta-endorphin metabolism, haloperidol and chlorpromazine were perfused via Alzet minipumps into male Sprague-Dawley rats for 8 days. Crude twice-washed membranes, purified synaptic plasma membranes and Golgi-enriched membranes, respectively, were isolated from rat brains and time course incubated with beta-endorphin. All samples were analyzed by high resolution, reversed-phase high performance liquid chromatography. The half-lives of beta-endorphin for animals treated with haloperidol or chlorpromazine were not statistically different from control animals at the crude washed membranes. At the purified synaptic plasma membranes, however, the half-lives of beta-endorphin from haloperidol (t 1/2 = 45.1 min)- and chlorpromazine (t1/2 = 47.0 min)-treated animals were significantly decreased as compared to the control animals (t1/2 = 78.0 min). The half-life of beta-endorphin at the Golgi-enriched membranes was increased for haloperidol (t1/2 = 112.3 min) and chlorpromazine (t1/2 = 103.0 min)-treated animals when compared to control animals (t1/2 = 80.2 min). The findings indicate a differential effect of the dopamine receptor antagonists haloperidol and chlorpromazine on the extracellular fate at the synaptic plasma membranes of beta-endorphin and the intracellular processing at the Golgi-enriched membranes in vitro.