Neuropeptide, detection

Biosynthesis. Two closely related genes encode the three mammalian tachykinins. The preprotachykinin A gene encodes both substance P and substance K, while the preprotachykinin B gene encodes neuromedin K (45—47). The active sequences are flanked by the usual double-basic amino acid residues, and the carboxy-terrninal amino acid is a glycine residue which is decarboxylated to an amide. As with most neuropeptide precursors, intermediates in peptide processing can be detected, but their biological activities are not clear (ca 1994). 

The endogenous release of the potent vasoconstrictor neuropeptide Y (NPY) is increased during sepsis and the highest levels are detected in patients with shock (A8). NPY is a 36-amino-acid peptide belonging to the pancreatic polypeptide family of neuroendocrine peptides (T2). It is one of the most abundant peptides present in the brain and is widely expressed by neurons in the central and peripheral nervous systems as well as the adrenal medulla (A3). NPY coexists with norepinephrine in peripheral sympathetic nerves and is released together with norepinephrine (LI9, W14). NPY causes direct vasoconstriction of cerebral, coronary, and mesenteric arteries and also potentiates norepinephrine-induced vasoconstriction in these arterial beds (T8). It appears that vasoconstriction caused by NPY does not counterbalance the vasodilatator effects of substance P in patients with sepsis. The properties of vasodilatation and smooth muscle contraction of substance P are well known (14), but because of the morphological distribution and the neuroendocrine effects a possible stress hormone function for substance P was also advocated (J7). Substance P, which is a potent vasodilatator agent and has an innervation pathway similar to that of NPY, shows a low plasma concentration in septic patients with and without shock (A8). 

Dirks RW, van Gijlswijk RP, Tullis RH et al (1990) Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides. J Flistochem Cytochem 38 467 173... 

The study of peptidergic neurons requires a number of special tools. These tools include methods to detect the neuropeptides both in cells and after release, the enzymes specific to their biosynthesis and their cognate receptors. Since the actions of peptides require secretion, measurements of cell content (e.g. immunostaining) can be deceptive, with a decrease in content reflecting increased release. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

Figure 2.15. Schematic of a quadrupole analyzer, (a) A hyperbolic cross-section (b) cross-section of cylindrical rods (c) the operating principle of a quadrupole mass filter. The x-direction pair of rods acts like a high pass filter so ion C (with low m/z) is not allowed through, and the y-direction pair of rods acts like a low pass filter and takes care of ion A (with high m/z). Only ion B having an m/z in the stable range is allowed through the quadrupole mass filter for subsequent detection. Reprinted from A. Westman-Brinkmalm and G. Brinkmalm (2002). In Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research, J. Silberring and R. Ekman (eds.) New York John Wiley Sons, 47-105. With permission of John Wiley Sons, Inc.

The molecular assays in Clk"mAic2As bom fide rhythms with a predominant effect on circadian rhythm amplitude and no more than a modest effect on phase or period. With circadian per and tim enhancers, we observed reduced enhancer activity and a reduced cycling amplitude in a Clk" background, consistent with the role of Clk in regulating these enhancers. Nonetheless, the phase of oscillating bioluminescence is similar to that of wild-type flies. The presence of molecular rhythms contrasts with the absence of detectable behavioural rhythms. We favour the notion that this reflects a level or amplitude reduction below a critical threshold for behavioural rhythmicity. The absence of anticipation of light—dark transitions makes it very unlikely that an effect restricted to the lateral neurons — the absence of the neuropeptide PDF, for example — is primarily responsible for the behavioural phenotypes. This is also because LD behavioural rhythms are largely normal in flies devoid of PDF or the pacemaker lateral neurons (Renn et al 1999). However, we cannot exclude the possibility of selective effects of Clk" on other behaviourally relevant neurons. 

Again, the physiological impact of this neuropeptide has to be confirmed in a proper animal model. Indeed, under both basal and stressful conditions, more AVP mRNA was detectable in the PVN of hyper-anxious HAB than LAB... 

The H3 receptor was initially detected as an autoreceptor controlling histamine synthesis and release in brain [22]. Thereafter it was shown to inhibit presynaptically the release of other monoamines in brain and peripheral tissues as well as of neuropeptides from unmyelinated C-fibers [23],... 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

McCormick J., Lim I. and Nichols R. (1999) Neuropeptide precursor processing detected by triple immunolabeling. Cell Tissue Res. 297, 197-202. 

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

Numerous tests carried out in insects with antibodies raised against mammalian neuropeptides revealed reaction products within and outside of the nervous system. Conversely, certain neuropeptides first identified in invertebrates were shown to occur also in mammals and other vertebrates. These commonalities are indicative of a long evolutionary history, as well as a wide distribution of active neuropeptides in neural and non-neural tissues. One of the new insights gained from the use of multiple antisera and carefully conducted specificity tests was the immunocytochemical detection in the insect brain of molecules closely resembling mammalian ACTH, prolactin, and insulin,. The localization of these substances suggests a neurotransmitterlike or neuromodulatory rather than a hormonal role. 

Bioassays commonly provide the initial means of detecting neuropeptide activity. The factor(s) responsible for this activity are assigned names reflecting the functions detected with the bioassay (e.g., adipoianetic hormone, AKH diuretic hormone, DH eclosion hormone, EH pheromone biosynthesis activating neuropeptide, PBAN prothoracicotropic hormone, PTTH and so forth). As more of these factors are discovered and characterized, not only are biologic ... 

---