Clinical value of pancreatitis-associated protein in acute pancreatitis

OBJECTIVES: Pancreatitis-associated protein (PAP) is a secretory protein that is overexpressed by the pancreas during acute pancreatitis. This study was carried out to assess the clinical value of PAP in acute pancreatitis, particularly its ability to indicate the severity of the disease. METHODS: Twenty-one healthy subjects, 58 patients with acute pancreatitis, and 20 patients with nonpancreatic acute abdomen were studied. In addition to serum PAP concentration, serum concentrations of amylase, lipase, and C-reactive protein (CRP) were measured at admission and, in patients with acute pancreatitis, during the following 5 days. RESULTS: On admission, serum PAP concentrations were abnormally high in 46 of the 58 patients with acute pancreatitis (79%); serum amylase, in 57 patients (98%); serum lipase, in all patients (100%); and serum CRP, in 40 patients (69%). During the subsequent days of the study, PAP and CRP tended to increase, whereas amylase and lipase decreased. No significant differences in PAP or amylase and lipase concentrations were found between patients with mild pancreatitis and those with severe pancreatitis during the entire study period, whereas from the third day to the sixth day, CRP concentrations were significantly higher in patients with severe pancreatitis than in those with mild pancreatitis. Among the 20 patients with nonpancreatic acute abdomen, PAP concentrations were abnormally high in 10 (50%), whereas amylase concentrations were abnormally high in five (25%), and lipase concentrations were high in two (10%). CONCLUSIONS: Our results indicate that the clinical value of PAP in acute pancreatitis is quite limited and, in particular, that PAP is not a useful marker for determining the severity of the disease.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Self-administration of neurokinin substance P into the ventromedial caudate-putamen in rats

There is evidence that the neurokinin substance P plays a role in learning and reinforcement processes. Reinforcing effects of substance P were found upon injection into several parts of the brain. The aim of the present study was to gauge possible reinforcing effects of microinjections of substance P into the ventromedial caudate-putamen in rats. Two different behavioral paradigms were employed. In the first experiment a two-compartment choice procedure was used and the rats could trigger substance P injections (500 pg per 5 nl injection volume) into the ventromedial caudate-putamen by entering one distinctive compartment. During the injection period, substance P-injected animals spent significantly more time in the drug-paired compartment than vehicle-injected controls. In the second experiment, nose-poking through a hole in one wall of the cage was used as the operant. Rats that could self-administer substance P (100 pg per 5 nl injection volume) into the ventromedial caudate-putamen emitted a significantly higher rate of operant responding on the first day of testing and a significantly lower rate on the third day compared to vehicle-injected animals. The experiments provide evidence that the administration of substance P into the ventromedial part of the caudate-putamen can have positive reinforcing effects, but that repeated injections can have aversive properties. These effects are discussed, firstly, with regard to the possible mechanisms of intrastriatal substance P on striatonigral and striatopallidal output systems and, secondly, with respect to their possible relevance in the study of the basal forebrain reinforcement system.     

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.     

Role of tachykinins in ozone-induced acute lung injury in guinea pigs

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3 group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.     

Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.     

Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body

Peripheral chemoreceptors are commonly thought to respond to hypoxia by releasing neurotransmitters from the type 1 cells of the carotid body; these molecules then bind to post-synaptic receptors on the carotid sinus nerve. The tachykinin substance P (SP) may act as an important neurotransmitter/neuromodulator in hypoxic chemotransmission in peripheral arterial chemoreceptors. In order to elucidate the role of SP in modulating hypoxic chemotransmission, we have used quantitative in situ hybridization histochemistry, to determine the effect of hypoxia on SP gene induction, and the localization of neurokinin 1 (NK-1) receptor mRNA in the carotid body and petrosal ganglia complex in rats at 21 days post-natal age. For comparison, we also determined: (1) the effect of hypoxia on tyrosine hydroxylase (TH) gene induction and (2) the localization of the mRNA encoding the D2-dopamine receptor. SP mRNA was not detected in the rat carotid body during normoxia and its expression was not induced after a 1 h of exposure to hypoxia (10% O2/90% N2), a stimulus that was sufficient to cause a significant increase (P < 0.01) in TH mRNA levels in the carotid body. Both SP and TH mRNAs were abundantly expressed in multiple cells in the petrosal and the jugular ganglia. However, these mRNAs were not co-localized and SP and TH mRNA levels were not affected by hypoxia in these ganglia. Although D2-dopamine receptor mRNA was abundantly expressed in the rat carotid body, we found no evidence of NK-1 receptor mRNA in the carotid body. In contrast, both NK-1 receptor mRNA and D2-dopamine receptor mRNA were present in petrosal ganglion cells. In the rat, SP does not appear to modulate hypoxic chemotransmission by being made in and released from type 1 cells in the carotid body, and neither does SP modulate the activity of type 1 cells by binding to NK-1 receptors on these cells.     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. L-tetrahydroisoquinoleic acid (Tic), L-fluorenylglycine (Flg), L-diphenylalanine (Dip), the diastereoisomers of L-1-Indanylglycine (Ing) and L-benz[f]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (delta ZPhe, delta EPhe, delta ZNal, ENal) and L-O,O'-dimethylphenylalanine (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S7 and S8 subsites, corresponding to residues Phe7 and Phe8 of substance P. According to the binding potencies of these substituted-SP analogues, the S7 binding subsite is smaller than the S8 subsite: the S7 subsite accepts only one aromatic nucleus, while the S8 can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S8 binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu6, Pro9]SP(6-11), discrepancies are observed between affinity (K1) and activity (EC50) values for IPs production. While a weak correlation between K1 and EC50 values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K1) and their potencies (EC50) for cAMP production (r = 0.97). The high potency (EC50) observed for "septide-like' molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC50 values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).     

Anxiolytic-like action of neurokinin substance P administered systemically or into the nucleus basalis magnocellularis region

There is evidence that the neurokinin substance P plays a role in neural mechanisms governing learning and reinforcement. Reinforcing and memory-promoting effects of substance P were found after it was injected into several parts of the brain and intraperitoneally. With regard to the close link between anxiety and memory processes for negative reinforcement learning, the aim of the present study was to gauge the effect of substance P on anxiety-related behaviors in the rat elevated plus-maze and social interaction test. Substance P was tested at injection sites where the neurokinin has been shown to promote learning and to serve as a reinforcer, namely in the periphery (after i.p. administration) and after injection into the nucleus basalis magnocellularis region. When administered i.p., substance P had a biphasic dose-response effect on behavior in the plus-maze with an anxiolytic-like action at 50 microg/kg and an anxiogenic-like one at 500 microg/kg. After unilateral microinjection into the nucleus basalis magnocellularis region, substance P (1 ng) was found to exert anxiolytic-like effects, because substance P-treated rats spent more time on the open arms of the plus-maze and showed an increase in time spent in social interaction. Furthermore, the anxiolytic effects of intrabasalis substance P were sequence-specific since injection of a compound with the inverse amino acid sequence of substance P (0.1 to 100 ng) did not influence anxiety parameters. These results show that substance P has anxiolytic-like properties in addition to its known promnestic and reinforcing effects, supporting the hypothesis of a close relationship between anxiety, memory and reinforcement processes.     

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.     

Expression of substance P (NK1) receptor mRNA in human nose

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(-)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00-140 microg ml(-1), for scopolamine 7.50-210 microg ml(-1) and for tropic acid 2.50-70.0 microg ml(-1).     

A possible involvement of oxidative lung injury in endotoxin-induced bronchial hyperresponsiveness to substance P in guinea pigs

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.     

Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation

1. Following induction of acute inflammation by intraarticular injection of kaolin and carrageenan into the knee joint in rats, there was a significant decrease in the withdrawal latency to radiant heat applied to the paw (i.e. heat hyperalgesia), an increased joint circumference and increased joint temperature. 2. A neurokinin1 (NK1) receptor antagonist (CP-99,994, 10 mM) had no effect on the paw withdrawal latency when it was administered spinally through a microdialysis fibre before the induction of inflammation. Pretreatment with a NK2 receptor antagonist (SR48968, 1 mM) administered spinally through the microdialysis fibre prevented the heat hyperalgesia from developing in the early stages of the inflammation. 3. Post-treatment through the microdialysis fibre with the NK1 receptor antagonist (0.01-10 mM) was effective in reversing the heat hyperalgesia. In contrast, post-treatment spinally with the NK2 receptor antagonist (0.01-1 mM) had no effect on the heat hyperalgesia. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia. 4. Pretreatment systemically with the NK1 receptor antagonist (30 mg kg-1) had no effect on the heat hyperalgesia or pain-related behaviour ratings where 0 is none and 5 is non weight bearing and complete avoidance of limb contact. Pretreatment with a NK2 receptor antagonist (10 mg kg-1) systemically prevented the heat hyperalgesia and pain-related behaviour ratings from developing in the early stages of the inflammation. The inactive stereoisomers of NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia. 5. Post-treatment systemically with either the NK1 (0.1-30 mg kg-1) or the NK2 (0.1-10 mg kg-1) receptor antagonist resulted in a dose-dependent reversal of the heat hyperalgesia. Pain-related behaviour ratings were reduced by post-treatment only with the NK1 receptor antagonist. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the behavioural responses. 6. Direct pretreatment of the knee joint with either the NK1 (30 mg) or the NK2 (10 mg) receptor antagonist prevented the heat hyperalgesia from developing without affecting joint swelling. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia. 7. There appears to be a differential role for the spinal tachykinin receptors in the development and maintenance of the heat hyperalgesia associated with acute joint inflammation. The NK2 receptors appear to be activated early in the development of the heat hyperalgesia and NK1 receptors are involved in the maintenance of the heat hyperalgesia. 8. Peripherally, both NK1 and NK2 receptors are involved in the development of heat hyperalgesia and pain-related behaviour ratings induced by acute inflammation.     

Release of lactate by the lung in acute lung injury

The pathogenesis of hyperlactatemia during sepsis is poorly understood. We have previously described an increase in lactate concentration across the lung in the dog during early endotoxemia. Accordingly, we sought to determine if the lung releases lactate in humans and what relation this has with lung injury. METHODS: We measured lactate concentrations across the lung and lung injury scores (LIS) in two groups of patients. Group 1 consisted of nine patients with acute lung injury (LIS > or = 2.0) and elevated lactate concentrations (> 2.0 mmol/L). Group 2 contained 12 patients with no acute lung injury (LIS scores < or = 1.5), with or without increased lactate concentrations. Simultaneous measurements of plasma lactate and blood gases were obtained from indwelling arterial and pulmonary artery catheters. Measurements of cardiac output were also obtained. Lactate measurements were done using a lactate analyzer (YSI; Yellow Springs, Ohio). RESULTS: For each patient with acute lung injury and hyperlactatemia, an arterial-venous lactate gradient existed demonstrating release of lactate by the lung. This gradient persisted after correction for changes in hemoconcentration across the lung. The lactate gradient across the lung was 0.4 +/- 0.2 mmol/L for group 1 vs 0.05 +/- 0.1 mmol/L for group 2 (p = 0.001). This corresponded to a mean pulmonary lactate flux of 231.3 +/- 211.3 vs 5.0 +/- 37.2 mmol/h (p = 0.001). The lactate flux and the arterial-venous lactate difference correlated with LIS both for the entire sample and for the subgroup with hyperlactatemia (r = 0.69, p < 0.01). Pulmonary lactate flux was not related to arterial lactate levels (r = 0.25). CONCLUSION: In patients with acute lung injury and hyperlactatemia, the lung is a major source of lactate and lactate flux correlates with LIS. This lactate flux could explain some of the hyperlactatemia seen in sepsis.     

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.     

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons

Agonist-induced endocytosis and recycling of G protein-coupled receptors contributes to desensitization and resensitization of the receptors. In this study, we have used fluorescence immunohistochemistry, confocal microscopy and digital image analysis to quantify the proportion of receptor in the cytoplasm and on the surfaces of nerve cells in the guinea-pig ileum. With these methods we examined the dynamics of internalization of the neurokinin 1 receptor in response to agonist, return of receptor to the cell membrane and its capacity to be re-internalized in response to further exposure to agonist. The basal level of neurokinin 1 receptor immunoreactivity in the cytoplasm was 12-15% of total cellular immunoreactivity. Concentration-response relations were generated for neurokinin 1 receptor internalization after incubation of isolated ileum with 10(-11) to 10(-6) M substance P at 4 degrees C and warming to 37 degrees C for 20 min. The threshold concentration for cytoplasmic receptor to exceed baseline was 10(-11) M and the proportion of receptor in the cytoplasm increased with increasing substance P concentration. The effect of two exposures to agonist was studied using 10(-8) M and 10(-6) M substance P. After equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C with no substance P, neurokinin 1 receptor immunoreactivity in the cytoplasm increased significantly from 12% to 36+/-3% for incubation with 10(-8) M and to 64+/-3% for 10(-6) M. When return of receptor to the surface was blocked with monensin (10(-5) M), 90% of the receptor was in the cytoplasm after 1 h at 37 degrees C following exposure to 10(-6) M substance P. After 60 min without substance P and no monensin, receptor in the cytoplasm decreased to 19+/-2% (10(-8) M) and 38+/-4% (10(-6) M). A second period of equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C, without substance P, resulted in a second wave of endocytosis; the fractions of receptor in the cytoplasm were 47+/-2% (10(-8) M) and 70 2% (10(-6) M). These results indicate that most of the receptors on the cell surface are available for internalization and that the receptors that return to the cell surface after endocytosis rapidly regain their ability to bind ligand and undergo endocytosis.