Krister Karlsson

Alteration in the brain content of substance P (1-7) during withdrawal in morphine-dependent rats

Previous studies have shown that substance P (SP) may modulate the abstinence reaction to opioid withdrawal. Its N-terminal fragment SP1-7 may inhibit the intensity of the withdrawal reactions in morphine dependent mice. This study was designed to determine whether the endogenous concentrations of the SP1-7 fragment in the brain are affected during naloxone-precipitated withdrawal in the male rat. The amounts of the peptide was assessed by a specific radioimmunoassay in extracts of discrete brain regions (including the cerebral cortex, hippocampus, hypothalamus, nucleus accumbens, striatum, substantia nigra, ventral tegmental area and the spinal cord) during morphine tolerance and withdrawal. The results indicated that the concentrations of SP1-7 were significantly elevated in the ventral tegmental area both in morphine tolerant rats and during naloxone-precipitated withdrawal. During morphine withdrawal significant increases in the peptide concentration were also observed in the hypothalamus and the spinal cord. It was concluded that the enhanced content of SP1-7 may also indicate the involvement of the SP system during opioid withdrawal in the rat. The enhanced production of SP1-7 may reflect an increased release and/or metabolism of SP, which, in turn, counteracts the withdrawal.

Substance P endopeptidase-like activity is altered in various regions of the rat central nervous system during morphine tolerance and withdrawal

In this study the level of a substance P endopeptidase (SPE)-like activity was measured in different regions of the rat central nervous system (CNS) after chronic administration of morphine. Male rats (200-220 g) were randomly divided into four groups. Two groups were injected (s.c.) with morphine (10 mg/kg) twice daily, whereas the other two received saline under identical conditions. After 8 days, when animals were completely tolerant to morphine, one of the morphine-treated groups and one group of saline-injected rats were given naloxone (s.c. 2 mg/kg). Withdrawal signs were observed and recorded. The enzyme activity was measured in extracts of the various CNS tissues by following the conversion of synthetic substance P (SP) to its N-terminal fragment SP(1-7) using a radioimmunoassay detecting this product. In discrete CNS areas including periaqueductal grey, spinal cord, substantia nigra and ventral tegmental area (VTA) a significant increase in enzyme activity was observed in the withdrawal group, while tolerant rats exhibited decreased SPE-like activity in the striatum (see Table 1). The enhanced enzyme activity during withdrawal is in agreement with our previous observation that the levels of SP(1-7) in rat brain are affected following naloxone precipitated withdrawal. In some tissues, including VTA, a correlation between the SPE-like activity and the intensity of the opioid abstinence was observed. Our result suggests that the elevated SPE-like activity is responsible for enhanced release of SP(1-7) in rats during morphine withdrawal, affirming a modulatory or regulative role of this enzyme in this state of opioid dependence.

Purification and characterization of substance P endopeptidase activities in the rat spinal cord

Two enzymes with substance P degrading activity were purified from the membrane bound fraction of the rat spinal cord. The purified enzymes were characterized with regard to biochemical and kinetic properties. One of the enzymes exhibited close similarity to neutral endopeptidase 24.11 (NEP, EC 3.4.24.11), while the other resembled a substance P converting endopeptidase (SPE), which has previously been identified and purified from human cerebrospinal fluid (CSF). Detergent treated spinal cord homogenates from male Sprague Dawley rats were purified by anion-exchange chromatography (DEAE-sepharose CL-6B), hydrophobic-interaction chromatography (phenyl-sepharose CL-4B) and molecular sieving (Sephadex G-50). Two fractions with enzymes differing in size were recovered and allowed for further purification to apparent homogeneity by ion-exchange chromatography and molecular sieving on a micro-purification system (SMART). The enzyme activities were monitored by following the conversion of synthetic substance P using a radioimmunoassay specific for the heptapeptide product, substance P (1-7). By SDS-polyacrylamide gel electrophoresis of the purified enzymes molecular weights of 43 and 70 kDa were estimated for the SPE-like and NEP-like activity, respectively. A K(m) of 5 microM was determined for the conversion of substance P to its (1-7) fragment by the SPE-like activity. Reversed-phase HPLC together with mass spectrometry permitted identification of all fragments released from substance P by the peptidases. The released fragments were for both enzymes identified as substance P (1-7), substance P (8-11), substance P (1-8), substance P (9-11). The NEP-like enzyme preparation also gave substance P (1-6) as a major product.

Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks.

Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin.

An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7‐Phe8 residues was purified from the membrane‐bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP‐hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy‐terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X‐100 in 20 mM Tris–HCl pH 7.8. After ion exchange chromatography (DEAE–Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G‐50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex® 75 HR 10/30) using a new preparative system, ÄKTATM‐purifier, controlled by UNICORN® software version 2.20. Copyright

Purification of substance P endopeptidase activity in the rat ventral tegemental area with the Akta-Purifier chromatographic system.

The new chromatographic system Akta-Purifier 10 (Amersham-Pharmacia Biotech), scaled for preparative HPLC, was used for the purification of Substance P (SP) endopeptidase activity in the ventral tegemental area (VTA) of the rat brain. SP endopeptidase previously identified and purified from human cerebrospinal fluid has been found to degrade the neuroactive peptide SP in a specific pattern. In this study we have recovered SP endopeptidase from the rat VTA following a purification scheme involving homogenization (ultrasonication) and extraction of the excised tissue, size-exclusion chromatography (Superdex 75 HR), and ion-exchange chromatography (Resource Q). In this way we were able to achieve a purification factor of almost 7,500, based on specific activity. The obtained SP endopeptidase activity, was then subjected to characterization with regard to inhibition profile. The enzyme activity was monitored by following the conversion of SP to its N-terminal fragment SP(1-7) using a radioimmunoassay, specific for the heptapeptide product. On basis of inhibition profile it was possible to discern two different SP endopeptidase-like activities, one sensitive toward the protease inhibitor phosphoramidon (preparation A), and another non-sensitive to phosphoramidon or captopril (preparation B). The molecular masses of preparations A and B, as derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were found to be 90,000 and 76,000, respectively. Our data suggest that the purified phosphoramidon sensitive endopeptidase activity may be an enzyme that plays a major role in the conversion of SP to its bioactive fragment SP(1-7) in the rat VTA. This is likely to be identical to the previously known neutral endopeptidase (EC 3.4.24.11). However, this study also demonstrates the existence of a distinct endopeptidase activity with properties in agreement with rat spinal cord SP endopeptidase. In the context of previously shown altered levels of SP(1-7) in the VTA during morphine withdrawal both purified enzyme activities may turn out to be responsible.