Anna Lesniak

Canine Olfactory Receptor Gene Polymorphism and Its Relation to Odor Detection Performance by Sniffer Dogs

The outstanding sensitivity of the canine olfactory system has been acknowledged by using sniffer dogs in military and civilian service for detection of a variety of odors. It is hypothesized that the canine olfactory ability is determined by polymorphisms in olfactory receptor (OR) genes. We investigated 5 OR genes for polymorphic sites which might affect the olfactory ability of service dogs in different fields of specific substance detection. All investigated OR DNA sequences proved to have allelic variants, the majority of which lead to protein sequence alteration. Homozygous individuals at 2 gene loci significantly differed in their detection skills from other genotypes. This suggests a role of specific alleles in odor detection and a linkage between single-nucleotide polymorphism and odor recognition efficiency.

A polymorphism in exon 2 of the δ-opioid receptor affects nociception in response to specific agonists and antagonists in mice selectively bred for high and low analgesia

&NA; This study searched for polymorphic sites in the murine &mgr;‐, &dgr;‐ and &kgr;‐opioid receptors that presumably influence pain perception. We employed two mouse lines divergently bred for high (HA, high analgesia line) and low (LA, low analgesia line) swim stress‐induced analgesia (SSIA). These mouse lines differ substantially in pain sensitivity, measured as hind paw withdrawal latency in the hot‐plate test. We found a novel C320T transition in exon 2 of the &dgr;‐opioid receptor gene, resulting in an A107V substitution in the first extracellular loop (EL1) of the peptide chain. Using hot‐plate and tail‐flick tests, we found a significant association between the genotype of this locus and basal nociception and SSIA magnitude. Moreover, this transition affects the pharmacological effects of two specific &dgr;‐opioid receptor ligands, the agonist SNC80 and the antagonist naltrindole. The impact of the C320T polymorphism on the magnitude of SSIA was partially mediated by endogenous opioids. The effectiveness of exogenous &dgr;‐opioid receptor ligands was greater in the HA than in the LA line, and was greater in C320C homozygotes than in C320T heterozygotes within each line. Our results indicate that the C320T polymorphism in the &dgr;‐opioid receptor gene is at least partly responsible for the divergent nociceptive thresholds in HA and LA mice under both basal and post‐stress conditions.

Association between the A107V substitution in the δ‐opioid receptors and ethanol drinking in mice selected for high and low analgesia

Experimental evidence suggests that endogenous opioids play an important role in the development of ethanol addiction. In this study, we employed two mouse lines divergently bred for opioid‐mediated stress‐induced analgesia. In comparison with HA (high analgesia line) mice, LA (low analgesia line) mice, having lower opioid receptor system activity, manifest enhanced basal as well as stress‐induced ethanol drinking. Here, we found that recently discovered C320T transition in exon 2 of the δ‐opioid receptor gene (EU446125.1), which results in an A107V substitution (ACA23171.1), leads to higher ethanol preference in CT mice compared with CC homozygotes. This genetic association is particularly evident under chronic mild stress (CMS) conditions. The interaction between stress and ethanol intake was significantly stronger in HA than in LA mice. Ethanol almost completely attenuated the pro‐depressive effect of CMS (assessed with the tail suspension test) in both the CC and CT genotypes in the HA line. In the LA mice, a lack of response to ethanol was observed in the CC genotype, whereas ethanol consumption strengthened depressive‐like behaviours in CT individuals. Our results suggest that constitutively active A107V substitution in δ‐opioid receptors may be involved in stress‐enhanced vulnerability to ethanol abuse and in the risk of ethanol dependence.

Biphalin protects against cognitive deficits in a mouse model of mild traumatic brain injury (mTBI)

Traumatic brain injury (TBI) is often a result of traffic accidents, contact sports or battlefield explosions. A mild form of traumatic brain injury (mTBI) is frequently underestimated, as the immediate physical symptoms decrease rapidly and conventional neuroimaging studies often do not show visible evidence of brain lesions. However, cognitive impairments persist for weeks, months or even years after the incident. Endogenous opioids were documented to play a role in thmodulation of mTBI pathology, whereas exogenous opioids were shown to possess neuroprotective properties. In the present study, biphalin, a dimeric enkephalin analog, improved cognitive performance in the Morris Water Maze and Novel Object Recognition tests in a mouse weight-drop model of mTBI. The effect of a single systemic injection of 10 mg/kg biphalin immediately after trauma was reversed by naltrexone, suggesting an opioid receptor-mediated mechanism. Biphalin also reduced cortical and hippocampal neurodegeneration, as shown by silver staining. Our data indicates that opioid receptor activation by biphalin may provide neuroprotection of post-traumatic neurodegeneration processes and may protect against memory impairments.

Chronic mild stress facilitates melanoma tumor growth in mouse lines selected for high and low stress-induced analgesia.

Abstract Both chronic stress conditions and hyperergic reaction to environmental stress are known to enhance cancer susceptibility. We described two mouse lines that displayed high (HA) and low (LA) swim stress-induced analgesia (SSIA) to investigate the relationship between inherited differences in sensitivity to stress and proneness to an increased growth rate of subcutaneously inoculated melanoma. These lines display several genetic and physiological differences, among which distinct sensitivity to mutagens and susceptibility to cancer are especially noticeable. High analgesic mice display high proneness both to stress and a rapid local spread of B16F0 melanoma. However, stress-resistant LA mice do not develop melanoma tumors after inoculation, or if so, tumors regress spontaneously. We found that the chronic mild stress (CMS) procedure leads to enhanced interlinear differences in melanoma susceptibility. Tumors developed faster in stress conditions in both lines. However, LA mice still displayed a tendency for spontaneous regression, and 50% of LA mice did not develop a tumor, even under stressed conditions. Moreover, we showed that chronic stress, but not tumor progression, induces depressive behavior, which may be an important clue in cancer therapy. Our results clearly indicate how the interaction between genetic susceptibility to stress and environmental stress determine the risk and progression of melanoma. To our knowledge, HA/LA mouse lines are the first animal models of distinct melanoma progression mediated by inherited differences in stress reactivity.

Biphalin preferentially recruits peripheral opioid receptors to facilitate analgesia in a mouse model of cancer pain - A comparison with morphine

The search for new drugs for cancer pain management has been a long-standing goal in basic and clinical research. Classical opioid drugs exert their primary antinociceptive effect upon activating opioid receptors located in the central nervous system. A substantial body of evidence points to the relevance of peripheral opioid receptors as potential targets for cancer pain treatment. Peptides showing limited blood-brain-barrier permeability promote peripheral analgesia in many pain models. In the present study we examined the peripheral and central analgesic effect of intravenously administered biphalin - a dimeric opioid peptide in a mouse skin cancer pain model, developed by an intraplantar inoculation of B16F0 melanoma cells. The effect of biphalin was compared with morphine - a golden standard in cancer pain management. Biphalin produced profound, dose-dependent and naloxone sensitive spinal analgesia. Additionally, the effect in the tumor-bearing paw was largely mediated by peripheral opioid receptors, as it was readily attenuated by the blood-brain-barrier-restricted opioid receptor antagonist - naloxone methiodide. On the contrary, morphine facilitated its analgesic effect primarily by activating spinal opioid receptors. Both drugs induced tolerance in B16F0 - implanted paws after chronic treatment, however biphalin as opposed to morphine, showed little decrease in its activity at the spinal level. Our results indicate that biphalin may be considered a future alternative drug in cancer pain treatment due to an enhanced local analgesic activity as well as lower tolerance liability compared with morphine.

Antinociceptive effect induced by a combination of opioid and neurotensin moieties vs. their hybrid peptide [Ile(9)]PK20 in an acute pain treatment in rodents.

Hybrid compounds are suggested to be a more effective remedy for treatment of various diseases than combination therapy, since the attenuation or total disappearance of side effects, typically induced by a single moiety, can be observed. This is of great importance, especially when we consider problems resulting from the use of opioid analgesics. However, although it seems that such compounds can be valuable therapeutic tools, the lack of conviction among the public as to the appropriateness of their use still remains; therefore patients are commonly treated with polypharmacy. Thus, in the presented paper we show a comparison of the antinociceptive effect between a novel opioid-neurotensin chimera called [Ile(9)]PK20 and a mixture of its structural elements, delivered intrathecally and systemically. Additionally, motor coordination was assessed in the rotarod test. The results clearly indicate that spinal administration of the examined compounds, resulted in a long-lasting, dose- and time-dependent antinociceptive effect. Although the mixture of both pharmacophores was found to be more active than [Ile(9)]PK20, motor impairments surfaced as a side effect. This in turn illustrates the advantageous use of hybrid structures over drug cocktails.

Importance of N-and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration

Abstract The heptapeptide SP1–7 (1, Arg1‐Pro2‐Lys3‐Pro4‐Gln5‐Gln6‐Phe7) is the major bioactive metabolite formed after proteolytic processing of the neuropeptide substance P (SP, Arg1‐Pro2‐Lys3‐Pro4‐Gln5‐Gln6‐Phe7‐Phe8‐Gly9‐Leu10‐Met11‐NH2). The heptapeptide 1 frequently exhibits opposite effects to those induced by SP, such as exerting antinociception, or attenuating thermal hyperalgesia and mechanical allodynia. The heptapeptide SP1–7 amide (2, Arg1‐Pro2‐Lys3‐Pro4‐Gln5‐Gln6‐Phe7‐NH2) is often more efficacious than 1 in experimental pain models. We have now assessed the anti‐allodynic outcome after systemic administration of 2 and a series of Ala‐substituted and truncated analogues of 2, in the spared nerve injury (SNI) mice model and the results obtained were correlated with in vitro plasma stability and permeability measurements. It is herein demonstrated that an intact Arg1 in SP1–7 amide analogues is fundamental for retaining a potent in vivo effect, while Lys3 of 2 is less important. A displacement with Ala1 or truncation rendered the peptide analogues either inactive or with a significantly attenuated in vivo activity. Thus, the pentapeptide SP3–7 amide (7, t1/2 = 11.1 min) proven to be the major metabolite of 2, demonstrated an in vivo effect itself although considerably less significant than 2 in the SNI model. Intraperitoneal administration of 2 in a low dose furnished the most powerful anti‐allodynic effect in the SNI model of all the analogous evaluated, despite a fast proteolysis of 2 in plasma (t1/2 = 6.4 min). It is concluded that not only the C‐terminal residue, that we previously demonstrated, but also the N‐terminal with its basic side chain, are important for achieving effective pain relief. This information is of value for the further design process aimed at identifying more drug‐like SP1–7 amide related peptidomimetics with pronounced anti‐allodynic effects. Graphical abstract Figure. No Caption available.

Impact of N-methylation of the substance P 1–7 amide on anti-allodynic effect in mice after peripheral administration

&NA; Substance P 1–7 (SP1–7, Arg1‐Pro2‐Lys3‐Pro4‐Gln5‐Gln6‐Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1–7 is having anti‐inflammatory, anti‐nociceptive and anti‐hyperalgesic effects in experimental models. Despite all encouraging properties of SP1–7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK‐1 receptor, the N‐terminal fragment SP1–7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1–7 amide 1 (Arg1‐Pro2‐Lys3‐Pro4‐Gln5‐Gln6‐Phe7‐NH2) was previously shown to be superior to the endogenous SP1–7 in all experimental pain models where the two compounds were compared. Herein, we report that N‐methylation scan of the backbone of the SP1–7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1–7 amide (3) and the [MeGln5]SP1–7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti‐allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N‐methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N‐terminal Arg1 and C‐terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti‐allodynic effect is emphasized. Notably, the three heptapeptides: the SP1–7 amide (1), the [MeLys3]SP1–7 amide (3) amide and the [MeGln5]SP1–7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain. Graphical abstract Figure. No caption available.

The effect of opioid agonists and antagonists on gastrointestinal motility in mice selected for high and low swim stress-induced analgesia.

The opioid system in the gastrointestinal (GI) tract plays an important physiological role, but is also responsible for the side effect of opioid drugs, including troublesome constipation in chronic pain treatment. The aim of this study was to characterize and validate a new mouse model to study the effects of opioid agonists and antagonists in the GI tract.