Increased negativity of interstitial fluid pressure in rat trachea in dextran anaphylaxis.

This study shows that, in rat trachea, dextran anaphylaxis is associated with increased negativity of interstitial fluid pressure (Pif) as measured with sharpened glass capillaries (tip diameter 3-7 microns) connected to a servo-controlled counterpressure system. Experiments were carried out in pentobarbital-anesthetized Wistar-M?ller rats. Pif in the control situation was -2.5 +/- 0.38 (SD) mmHg. The mean pressure in animals killed 2 min after initiation of the anaphylactic reaction by injection of 1 ml of 10% Dextran 70 in 0.9% NaCl was -10.3 +/- 2.6 mmHg. In another experimental series, interstitial fluid volume was measured after dextran administration but without inducing circulatory arrest. Interstitial fluid volume increased from 0.94 +/- 0.16 to 1.56 +/- 0.42 ml/g dry wt after 10 min to 1.57 +/- 0.30 and 1.10 +/- 0.27 ml/g dry wt after 30 and 60 min, respectively. The increased negativity in Pif in tracheal mucosa in the early phase of dextran anaphylaxis will markedly increase the transcapillary net filtration pressure in the initial phase of edema development.     

Fluorescent derivatives of bovine neurotensin 8–13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.     

Fluorescent derivatives of bovine neurotensin 8–13 fragment

Summary Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.     

Synthesis and biological studies of novel neurotensin(8-13) mimetics

Novel neurotensin (NT) (8–13) (Arg⁸-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³) mimetics 3, 4 were designed by adopting all intrinsic functional groups of the native neurotensin(8–13) and using a substituted indole as a template to mimic the pharmacophore of NT(8–13). Biological studies at subtype 1 of the NT receptor showed that 3 has a 55 and 580 nM binding affinity at rat and human neurotensin receptors, respectively. As a comparison, compounds 5 and 6 were also synthesized. The binding difference between 3, 4 and 5, 6 argues the importance of the carboxylic group in achieving higher potency NT(8–13) mimetics.     

Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.     

Preparation and receptor binding affinities of cyclic C-terminal neurotensin (8-13) and (9-13) analogues.

Cyclic analogues of neurotensin (NT) C-terminal fragments NT(8-13) and NT(9-13) were produced via intramolecular nucleophilic substitution of the Tyr(11) phenoxide anion on a 6-bromohexanoyl side chain substituted at position 8 or 9 and tested for NT receptor binding affinity.     

Long-term lowering of tumour interstitial fluid pressure reduces Ki-67 expression

High tumour interstitial fluid pressure (TIFP) is a characteristic of most solid tumours. Recent data give first evidence that mechanical stretch induced by TIFP triggers proliferation in solid tumours. In the present study we compared two protocols of TIFP reduction on the expression of the tumour proliferation marker Ki-67: (a) short-term lowering of TIFP by a singular puncture and (b) long-term lowering of TIFP by catheterization. Utilizing two experimental tumours (A431, A549) it was found that the TIFP broke down rapidly after a singular puncture but recovered within 6h. In case of permanent catheterization no TIFP recovery was observed. After 24h tumours were excised and stained against the proliferation marker Ki-67. While a singular puncture had no effect catheterized tumours showed a significant decrease in Ki-67 expression. Our data suggest that long-term lowering of TIFP is required to reduce tumour proliferation.     

Synthesis of neurotensin(9-13) analogues exhibiting enhanced human neurotensin receptor binding affinities

Recent evidence is consistent with neurotensin (NT)(8-13) adopting a Type I beta-turn conformation while binding the NT receptor, which would place the cationic side-chains of Arg(8) and Arg(9) in close proximity. This was the basis for the design, synthesis and analysis of truncated NT(9-13) analogues 1-5 with dicationic position 9 side-chains to emulate the functions of the 8 and 9 side-chains of NT(8-13).     

Topography of the neurotensin (NT)(8-9) binding site of human NT receptor-1 probed with NT(8-13) analogs.

A series of neurotensin (NT)(8-13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non-natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor-1 (hNTR-1). The modifications were designed to probe specific steric and electrostatic requirements in the N-terminal cationic region of NT(8-13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non-natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the alpha-amino acid carbon. Substitution of Arg8 with N(G)-alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8-13) to the hNTR-1. Position 8 incorporation of Hlys produced the most favorable primary amine side-chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8-13), in which DAB is 2,4-diaminobutyric acid, was also prepared and tested as the shorter side-chain of DAB is known to be favored in position 9 of NT(8-13). This analog maintained 60% of NT(8-13) binding affinity making it the most favored des-guanidinium-containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR-1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8-13) and related analogs.     

A novel tetrabranched neurotensin(8-13) cyclam derivative: synthesis, 64Cu-labeling and biological evaluation

New macrocyclic 1,4,8,11-tetraazacyclotetradecane (cyclam) derivatives with 1, 2 and 4 neurotensin(8-13) units 4, 5 and 7 have been synthesized. Compounds 4 and 5 were prepared by the reaction of non-stabilized neurotensin(8-13) and cyclamtetrapropionic acid 2 using 1-ethyl-3-(3-dimethylaminocarbonyl)carbodiimide-hydrochloride and N-hydroxysulfosuccinimide. The tetrameric compound 7 was synthesized by Michael addition of neurotensin(8-13) acrylamide 6 and cyclam 1. The copper(II) complexation behavior of 4, 5 and 7 was investigated by UV/visible spectrophotometry and shows that the metal center resides inside the N4 chromophore with additional apical interactions established with pendant arms. The novel tetrabranched NT(8-13) cyclam 7 with nanomolar neurotensin receptor 1 binding affinity was efficiently radiolabeled with ⁶⁴Cu under mild conditions. ⁶⁴Cu ⊂ 7 showed slow transchelation in the presence of a large amount of cyclam as competing ligand, while it completely remains intact in the presence of EDTA. The in vivo behavior of ⁶⁴Cu ⊂ 7 was studied in rats and mice. The metabolic stability in rodent models was high with a half-life of intact ⁶⁴Cu ⊂ 7 in plasma of 34 min in rats and 60 min in the mice, respectively. The binding affinity was high enough to demonstrate in vivo binding of ⁶⁴Cu ⊂ 7 to NTR1 overexpressing HT-29 tumor xenotransplants in nude mice. Regarding elimination, ⁶⁴Cu ⊂ 7 showed a substantial renal and reticuloendothelial accumulation. On the other hand, metabolization of the compound in vivo with a resulting metabolite—postulated to be the ⁶⁴Cu-cyclam-tetraarginine complex—also showed long retention in the circulating blood, preventing a better contrast of tumor imaging.     

Precursor forms of neurotensin (NT) in cat: processing with pepsin yields NT-(3-13) and NT-(4-13)

Basic proteins present in 0.1 N HCl extracts of feline CNS and intestine were found to liberate immunoreactive neurotensin (iNT) when treated with hog pepsin. These protein substrates were separated using Sephadex G-25, Sephadex G-75 and reverse-phase HPLC. In a calibrated SDS-polyacrylamide gel electrophoresis system, the major substrate from cat ileum exhibited a molecular weight of ca 16 kDa and minor substrates were observed at 30, 40 and 65 kDa. As shown previously for synthetic NT, pepsin-treatment of feline ileal NT converted it into the fully immunoreactive NT-(4-13) fragment (yield, 95%). When treated with pepsin, the partially purified ileal substrates gave rise to 4 immunoreactive peptides, one of which (ca 15% of total) eluted with the same retention time as NT-(4-13) while the major peptide formed (ca 40% of total) eluted near to the position of NT-(3-13). Both these products reacted equally well with two different antisera towards the C-terminal 5- and 8-residues of NT and were not recognized by an N-terminal antiserum. Experiments using various proteases demonstrated that the NT-related sequence(s) were located internally in each substrate and suggested that they were bounded by double basic residues. Substrate activity in isotonic homogenates of feline spinal cord, brain, adrenal and ileum cosedimented with iNT during equilibrium centrifugation, apparently in association with vesicle and/or synaptosomal particles. These findings indicate that basic proteins, colocalized with NT in vesicle-like particles of CNS, adrenals and ileum, could serve as precursors to this peptide, being liberated by pepsin-related enzyme(s).     

Regulation of extracellular volume and interstitial fluid pressure in rat bone marrow

The volume and fluid pressure characteristics of the intact bone marrow is incompletely understood. We used microspheres and lipoproteins for measurements of intravascular volume (IVV) and EDTA for interstitial fluid volume (IFV) within the rat bone marrow. Interstitial fluid pressure (IFP) was determined with micropipettes connected to a servo-controlled counter-pressure system. Both the microspheres and the lipoproteins yielded estimates of IVV of approximately 1 ml/100 g. After a brief reactive hyperemia, IVV increased to 2.5 ml/100 g, whereas IFV decreased with approximately 1.5 ml/100 g, so that total extracellular volume did not change. Baseline bone marrow IFP was 9.7 mmHg. The hyperemia led to a transient twofold increase in IFP, whereas a marked blood loss decreased IFP by almost one-half. These novel data suggest that extracellular volume and IFP within the bone marrow can be measured with tracer methods and the micropuncture technique. The responses of IVV, IFV, and IFP during changes in blood flow to the bone marrow suggest a tight regulation and are thus compatible with those for a low-compliant tissue.     

Relationship between interstitial fluid volume and pressure (compliance) in hypothyroid rats

There is clinical and experimental evidence that lack of thyroid hormones may affect the composition and structure of the interstitium. This can influence the relationship between volume and pressure during changes in hydration. Hypothyrosis was induced in rats by thyroidectomy 8 wk before the experiments. Overhydration was induced by infusion of acetated Ringer, 5, 10, and 20% of the body weight, while fluid was withdrawn by peritoneal dialysis with hypertonic glucose. Interstitial fluid pressure (P(i)) in euvolemia (euvolemic control situation) and experimental situation was measured with micropipettes connected to a servocontrolled counterpressure system. The corresponding interstitial fluid volume (V(i)) was found as the difference between extracellular fluid volume measured as the distribution volume of (51)Cr-labeled EDTA and plasma volume measured using (125)I-labeled human serum albumin. In euvolemia, V(i) was similar or lower in the skin and higher in skeletal muscle of hypothyroid than in euthyroid control rats, whereas the corresponding P(i) was higher in all tissues. During overhydration, P(i) rose to the same absolute level in both types of rats, whereas during peritoneal dialysis there was a linear relationship between volume and pressure in all tissues and types of rats. Interstitial compliance (C(i)), calculated as the inverse value of the slope of the curve relating changes in volume and pressure in dehydration, did not differ significantly in the hindlimb skin of hypothyroid and euthyroid rats. However, in skeletal muscle, C(i) was 1.3 and 2.0 ml. 100 g(-1). mmHg(-1) in hypothyroid and euthyroid rats (P < 0.01), with corresponding numbers for the back skin of 2.7 and 5.0 ml. 100 g(-1). mmHg(-1) (P < 0.01). These experiments suggest that lack of thyroid hormones in rats changes the interstitial matrix, again leading to reduced C(i) and reduced ability to mobilize fluid from the interstitium.     

Characterization of neurotensin(6-13) from an hepatic fibrolamellar carcinoma.

Neurotensin(6-13) has been isolated and sequenced as the major form of neurotensin-like immunoreactivity (NTLI) in a human hepatic fibrolamellar carcinoma. Circulating NTLI in the patient, especially C-terminal, was very high. In additional studies, NT(6-13) was synthesized and compared with the purified tumor NTLI by HPLC analysis and by testing stability in plasma in vitro. These methods confirmed that the tumor NTLI was identical to NT(6-13). Since the metabolic clearance rate of synthetic NT(6-13) in sheep was 30-fold higher than NT(1-13), it suggests that the elevated plasma levels are the result of impaired clearance and/or markedly elevated production.     

Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation.

Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 +/- 0.03 (n = 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n = 5) was 18.8 +/- 0.6 mmHg with a corresponding pressure in plasma of 22 +/- 1.5 mmHg, whereas after overhydration (n = 5) these pressures fell to 9.8 +/- 0.4 and 11.9 +/- 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1 beta, IL-6, and TNF-alpha in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1 beta and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.     

Monitoring neurotensin[8-13] degradation in human and rat serum utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We have developed a novel method for rapid and empirical mapping of the protein interaction domain using a unique and atypical PCR-based amplification and a conventional yeast two-hybrid system. The modified PCR, designated as PASA-PCR, enables preferential amplification of the shortest amplicon from a complex expression library. PASA-PCR consists of reiterative cycles of denaturation of template DNAs and extremely abbreviated annealing/extension of primers to prevent their complete extension in a single cycle, followed by conventional amplification cycles. In PASA-PCR, the shortest (ranging from 400 to 1000 bp) amplicon is amplified almost exclusively from templates of various amplicon sizes. In addition, the frequency of in vitro recombination can be increased using low cooling rates (<0.5 degrees C/s) between the denaturation and annealing/extension steps, which was helpful in generating precisely trimmed protein-coding regions. Identification of Spc19-binding region of Spc34, which is a component of yeast's spindle pole body, was achieved by a combination of the yeast two-hybrid system and PASA-PCR.     

The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes.

TMB-8 inhibited respiration of rat thymocytes and rat liver mitochondria, probably by inhibition of NADH dehydrogenase. TMB-8 markedly decreased both the cellular ATP concentration and the mitochondrial membrane potential in situ in thymocytes. These effects occurred at, or well below, the concentrations used in other systems to investigate the role of intracellular calcium pools in signalling events. We conclude that caution should be exercised in the interpretation of the effects of TMB-8.     

大鼠侧脑室注射神经降压素对血压的作用

雄性Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠,用乌拉坦腹腔麻醉,在侧脑室注射神经降压素（ＮＴ）（１０,２０μｇ）可引起血压升高或降低,心率减慢,预先ｉｃｖ ａ１受体阻断剂哌唑嗪,可阻断ＮＴ的中枢升压反应,预先ｉｃｖ Ｍ受体阻断剂硫酸阿托品,可阻断ＮＴ的中枢降压反应,预先ｉｃｖ Ｈ１受体阻断剂扑尔敏或Ｈ２受体阻断剂甲氰咪胍,对ＮＴ的中枢心血管效应均无明显影响。实验结果表明：脑中ＮＴ升高可使血压升高或降低;在