Effects of endokinin A/B, endokinin C/D, and endomorphin-1 on the regulation of mean arterial blood pressure in rats

Endokinins are four novel human tachykinins, including endokinins A (EKA), B (EKB), C (EKC), and D (EKD). Endokinin A/B (EKA/B) is the common C-terminal decapeptide in EKA and EKB, while endokinin C/D (EKC/D) is the common C-terminal duodecapeptide in EKC and EKD. In this study, we attempted to investigate the interactions between EKA/B, EKC/D, and endomorphin-1 (EM-1) on the depressor effect at peripheral level. The effects of EKA/B produced a U-shaped curve. The maximal effect was caused by 10 nmol/kg. EKC/D and EM-1 showed a dose-dependent relationship. Co-administration of EKA/B (0.1, 1, 10 nmol/kg) with EM-1 produced effects similar to those of EKA/B alone but slightly lower. Co-injection of EKA/B (100 nmol/kg) with EM-1 caused an effect stronger than any separate injection. Co-administration of EKC/D (10 nmol/kg) with EM-1 (30 nmol/kg) caused a depressor effect, which was one of the tradeoffs of EM-1 and EKC/D. Mechanism studies showed that SR140333B could block the depressor effects of EKA/B, EKC/D, EM-1, EKA/B + EM-1, and EKC/D + EM-1; SR48968C could block EM-1, EKA/B, EKC/D, and EKC/D + EM-1 and partially block EKA/B + EM-1; SR142801 could block EM-1, EKC/D, and EKC/D + EM-1 and partially block EKA/B and EKA/B + EM-1; naloxone could block EM-1, EKC/D, and EKC/D + EM-1 and partially block EKA/B and EKA/B + EM-1. Pretreatment with NG-nitro-l-arginine methyl ester partially decreased depressor intensity and half-recovery time of EKA/B and EKC/D.     

Effects of endokinin A/B and endokinin C/D on the antinociception properties of hemopressin in mice

The current study evaluated the effects of hemopressin (HP) on pain modulation by endokinin A/B (EKA/B) and endokinin C/D (EKC/D) at the supraspinal level in mice. Intracerebroventricular administration of HP (10 nmol) fully antagonized the hyperalgesia induced by EKA/B (10, 30, and 100 pmol), and induced a dose-dependent potent analgesic effect. HP at different concentrations (10 pmol, 100 pmol, and 1 nmol) showed varying effects on the analgesic effect of EKA/B (3 nmol). HP extended the duration of the analgesic effect of EKC/D (3 nmol). Moreover, HP at different concentrations (10 pmol, 5 pmol, 1 pmol, and 100 fmol) co-administered with EKC/D (30 pmol) induced significant analgesia at two different time points: 5 min and 50 min. To investigate the antinociceptive mechanism, we used SR140333B and SR142801. HP (1 pmol) potentiated the analgesic effect of SR140333B (100 pmol) + EKA/B (30 pmol) in 5–10 min, while HP (100 pmol) had no effect in the analgesia induced by SR140333B (3 nmol) + EKA/B (3 nmol). HP (1 nmol) fully inhibited the analgesic effect of SR140333B (3 nmol) + EKC/D (3 nmol) or SR142801 (3 nmol) + EKC/D (3 nmol). HP (1 pmol) weakened the analgesic effect of SR142801 (100 pmol) + EKA/B (30 pmol), but HP (100 pmol) strengthened the analgesic effect of SR142801 (3 nmol) + EKA/B (3 nmol). These findings may pave the way for a new strategy on investigating the interaction between tachykinins and opioids on pain modulation.     

Experimental data regarding the implications of certain minimum structure enkephalin-like peptides in nociceptive processing.

The aim of this study was to investigate the importance of the amino acidic sequence at N-terminal end of certain minimum structure enkephalin-like peptides for the analgesic activity. Different groups of mice or rats were treated with 1) L-tyrosine (i.p. 200 mg/kg), 2) Tyr-Phe (i.t. 0.5 mg/rat), 3) Tyr-Pro-Phe (i.t. 0.5 mg/rat), 4) Gly-Tyr (i.t. 0.5 mg/rat), 5) Tyr-Gly-Gly (i.t. 0.5 mg/rat). Different tests were utilized to evaluate the antinociceptive effect of the substances tested: thermal nociception (hot plate test, plantar test), mechanical nociception (analgesymeter test). Tyr-Pro-Phe, Tyr-Gly-Gly, Tyr-Phe, but not Gly-Tyr, elicited analgesic activity. So, the presumption made in the case of atypical opioid peptides that opioid-like activity in case of peptides presumes a tyrosine residue at the N-terminal sequence, applies for shorter peptides. It appears also that minimal structure brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opioids, can provide better affinity for the opioid receptors and stronger analgesic activity. The inhibition of their analgesic effect by previous administration of naloxone proves that this effect is mediated through the endogenous opioid system.     

Brain processing of hemorphin-7 peptides in various subcellular fractions from rats

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.     

Pharmacological characterisation of spider antimicrobial peptides

Most spider antimicrobial peptides share a common mechanism of membrane permeabilisation and the innate immune systems of the pathogen. In this review, we present recent accounts of the application at the preclinical level that should be tried and the range of bioactivities and their particular structure can be harnessed for molecular engineering applications and in drug design. Structural analyses such as amino acid sequence and circular dichroism are described. Conductance measurements and pharmacological studies of the action on the inner or outer membranes of anti-microbe will reveal more about the mode of action of the antimicrobial peptides of spider.     

Pharmacological characterization of the ghrelin receptor mediating its inhibitory action on inflammatory pain in rats

Recent research suggests a role for ghrelin in the modulation of inflammatory disorders. However, the type of ghrelin receptor (GHS-R) involved in both the anti-inflammatory and anti-hyperalgesic actions of ghrelin remains to be characterized. In this study, we examined whether the inhibitory effect of ghrelin in the development of hyperalgesia and edema induced by intraplantar carrageenan administration depends on an interaction with GHS-R1a. Both central (1 nmol/rat, i.c.v.) and peripheral (40 nmol/kg, i.p.) administration of the selective GHS-R1a agonist EP1572 had no effect on carrageenan-induced hyperalgesia measured by Randall–Selitto test and paw edema. Furthermore, pre-treatment with the selective GHS-R1a antagonist, d-lys³-GHRP-6 (3 nmol/rat, i.c.v.) failed to prevent the anti-hyperalgesic and anti-inflammatory effects exerted by central ghrelin administration (1 nmol/rat), thus indicating that the type 1a GHS-R is not involved in these peptide activities. Accordingly, both central (1 and 2 nmol/rat, i.c.v.) and peripheral (40 and 80 nmol/kg, i.p.) administration of desacyl-ghrelin (DAG), which did not bind GHS-R1a, induced a significant reduction of the hyperalgesic and edematous activities of carrageenan. In conclusion, we have shown for the first time that DAG shares with ghrelin an inhibitory role in the development of hyperalgesia, as well as the paw edema induced by carrageenan and that a ghrelin receptor different from type 1a is involved in the anti-inflammatory activities of the peptide.     

Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides. Implications for control of viral processing

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.     

Spinal processing: anatomy and physiology of spinal nociceptive mechanisms

The processing of nociceptive input that occurs at the spinal level represents the first stage of effective control over its access to higher regions of the central nervous system. Recent developments in both the anatomy and physiology of nociceptive processing pathways at this level are beginning to yield an integrated understanding of structure and function. Most small afferent axons terminate in the more superficial laminae of dorsal horn, but technical difficulties have, until recently, prevented analysis of the functional properties of identified small fibres. A direct input of nociceptive afferents on to particular dorsal horn neurons is difficult to establish in view of the slow impulse conduction in these fibres and the small size of target neurons in the substantia gelatinosa. The small cells themselves are being analysed for relations between structure and function, using physiological, intracellular staining and immunocytochemical techniques to characterize their properties. They appear to be a highly heterogeneous population with many sub-classes, whether typed according to the transmitter they contain, e.g. enkephalin, to their physiological responses: whether excitatory or inhibitory to nociceptive and other inputs, or to both. The multireceptive neurons that project out of the dorsal horn toward supraspinal regions are, in general, located in deeper laminae and are likely to receive nociceptive information through polysynaptic pathways. The nocireceptive neurons in lamina I, which receive exclusively nociceptive inputs from myelinated and non-myelinated afferents project, at least in part, to thalamic and brain stem regions. Polysynaptic nociceptive pathways in dorsal horn may be subject to different controls from neurons in laminae I and II. Tonic descending inhibition is operative on the former and it is becoming clearly established that descending systems such as those containing noradrenaline, can regulate the access of nociceptive information to higher levels. The mechanisms of such descending controls and the importance of their interaction with segmental control systems, such as those involving the dynorphin opioids, are just beginning to be understood. Many somatosensory neurons in dorsal horn, both the large cells, some of which project supraspinally, and the small cells of superficial laminae, receive convergent nociceptive and non-nociceptive inputs. Although solely nociresponsive neurons are clearly likely to fill a role in the processing and signalling of pain in the conscious central nervous system, the way in which such useful specificity could be conveyed by multireceptive neurons is difficult to appreciate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological effect of beta toxin of Clostridium perfringens type C on rats

The biological effect of purified beta toxin of Clostridium perfringens type C in vivo was investigated. After intravenous injection of the purified beta toxin into rats, a rise in blood pressure and a simultaneous fall in heart rate were observed. After the blood pressure reached a maximum, the heart rate recovered gradually, and electrocardiographic and respiratory changes began. The rise in blood pressure induced by beta toxin tended to be proportional to the amount of toxin. The latent period between the injection of toxin and the onset of the increase, and also the time between the injection and the maximum pressure induced by the toxin decreased with increasing concentration of the toxin. A good correlation was found between the factor producing the rise in blood pressure and beta toxin. Alpha adrenergic and ganglionic blocking agents reduced blood pressure levels elevated by beta toxin. The data suggest that the toxin causes a release of catecholamines, and that the increase in blood pressure was induced by released catecholamines.     

Individual characteristics of the nociceptive sensitivity of rats during the formation of a negative emotional reaction

Changes in dynamics of nociceptive sensitivity in male rats of Wistar strain were studied during formation of negative emotional reaction. The procedure of blood's taking from the tail after its tip's amputation was used as negative emotional factor. The animals were divided into six groups by the criterion of individual properties of nociceptive sensitivity dynamics. On the whole it was found to decrease. Besides, one more group (the seventh group) was found with profound hypoalgesia caused by action of the negative emotional factor. It was also shown that formation of negative emotional reaction in two groups was not accompanied by changes of nociception. The data suggest a selective influence of negative emotional factor on nociceptive sensitivity.     

Democratic organization of the thalamocortical neural ensembles in nociceptive signal processing

Acute pain is a warning protective sensation for any impending harm. However, chronic pain syndromes are often resistant diseases that may consume large amount of health care costs. It has been suggested by recent studies that pain perception may be formed in central neural networks via large-scale coding processes, which involves sensory, affective, and cognitive dimensions. Many central areas are involved in these processes, including structures from the spinal cord, the brain stem, the limbic system, to the cortices. Thus, chronic painful diseases may be the result of some abnormal coding within this network. A thorough investigation of coding mechanism of pain within the central neuromatrix will bring us great insight into the mechanisms responsible for the development of chronic pain, hence leading to novel therapeutic interventions for pain management.     

Trait anxiety affects the orofacial nociceptive response in rats

The aims of the present study were to assess the influence of: a) trait anxiety on orofacial pain; and b) orofacial pain on state anxiety. Forty-four rats were initially exposed to the free-exploratory paradigm for the evaluation of their anxiety profiles. In accordance to the parameter "Percentage of time in the novel side", the animals were considered as presenting high or low levels of trait anxiety when presenting values below the 1st quartile, or above the 3rd quartile, respectively. A week later, formalin-1.5% was injected into the upper lip of each animal. The behavioural nociceptive response, characterized by increased orofacial rubbing (OR), was quantified for 30 minutes, as follows: Total time OR (0-30 minutes: total pain), 1st phase OR (0-6 minutes: neurogenic pain), and 2nd phase OR (12-30 minutes: inflammatory pain). Immediately after this test, but still under the effect of formalin, the rats were submitted to the Elevated Plus-maze test (EPM). The results showed that the high trait anxiety individuals presented higher frequency of OR than the low trait anxiety ones, except during the neurogenic pain period. However, no correlation was found between OR frequency and levels of state anxiety presented on the EPM. In conclusion, the animals presenting higher anxiety profiles were the most susceptible to orofacial pain, nevertheless, orofacial pain did not influence state anxiety.     

Rapid determination of the thermal nociceptive threshold in diabetic rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli¹. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus². However, as stated by Bölcskei et al, determination of "pain" in animal models is challenging due to its subjective nature³. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity³. For instance, by using the hot-plate method of Ankier⁴, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 °C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures^3,5. As the recently described method of Bölcskei et al.⁶, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Almási et al⁵ and Bölcskei et al³. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.     

The determination of the nociceptive thresholds in freely moving rats

A simple method for determination of nociceptive thresholds in free-moving rats is put forward. The method is based on the registration of vocalization reaction caused by the feed through the lungs connected with the stimulator by thin wires electrodes of gradually increasing impulses of constant electric current. The examples of effect of morphin and naloxone on the nociceptive thresholds as well the alteration of thresholds during predatory aggression are given.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys⁵⁷-Arg⁷⁷). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.     

Bioactive dietary peptides and amino acids in inflammatory bowel disease

Inflammatory bowel disease (IBD), most commonly ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic inflammation of the gastrointestinal tract. Patients affected with IBD experience symptoms including abdominal pain, persistent diarrhea, rectal bleeding, and weight loss. There is no cure for IBD; thus treatments typically focus on preventing complications, inducing and maintaining remission, and improving quality of life. During IBD, dysregulation of the intestinal immune system leads to increased production of pro-inflammatory cytokines, such as TNF-α and IL-6, and recruitment of activated immune cells to the intestine, causing tissue damage and perpetuating the inflammatory response. Recent biological therapies targeting specific inflammatory cytokines or pathways, in particular TNF-α, have shown promise, but not all patients respond to treatment, and some individuals become intolerant to treatment over time. Dietary peptides and amino acids (AAs) have been shown to modulate intestinal immune functions and influence inflammatory responses, and may be useful as alternative or ancillary treatments in IBD. This review focuses on dietary interventions for IBD treatment, in particular the role of dietary peptides and AAs in reducing inflammation, oxidative stress, and apoptosis in the gut, as well as recent advances in the cellular mechanisms responsible for their anti-inflammatory activity.