Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective Compounds against Drug Resistant Clinical Isolates of E. coli.

Evolution-derived natural compounds have been inspirational for design of numerous pharmaceuticals, e.g., penicillins and tetracyclines. Herein, we present a bioinspired strategy to design peptide dendrimers for the effective therapy of E. coli infections where the selection of appropriate amino acids and the mode of their assembly are based on the information gained from research on membranolytic natural antimicrobial peptides (AMP's). On the molecular level two opposite effects were explored: the effect of multiple positive charges necessary for membrane disintegration was equilibrated by the anchoring role of tryptophanes. Indeed, a series of Trp-terminated dendrimers exhibited high potency against clinical isolates of antibiotic resistant ESBL E. coli strains, stability in human plasma along with very low hemo- and genotoxicity. Investigation of the underlying antimicrobial mechanism indicated that the dendrimers studied at minimal inhibitory concentration showed weak permeability toward membranes. Solid-state 2D NMR studies revealed their presence on and inside the model membranes. Therefore, their biological properties might be explained by targeting of extra- or intracellular receptors. Our results point to a new approach to design novel branched antimicrobials with high therapeutic index.

The role of allogenic keratin-derived dressing in wound healing in a mouse model.

Keratin is an interesting protein needed for wound healing and tissue recovery. We have recently proposed a new, simple and inexpensive method to obtain fur and hair keratin-derived biomaterials suitable for medical application. The aim of the study was to evaluate the role of the fur keratin-derived protein (FKDP) dressing in the allogenic full-thickness surgical skin wound model. The data obtained using scanning electron microscopy showed that employed processed biomaterial had higher surface porosity compared with control raw material. From the MTS test, it was found keratin biomaterial is not only toxic to the NIH/3T3 cell line (p < 0.05), but also enhances cell proliferation compared with the control. In vivo studies have shown keratin dressings are tissue biocompatible, accelerate wound closure and epithelialization to the statistically significant differences on day 5 (p < 0.05) in comparison to control wounds. Histological examination revealed, that in FKDP-treated wounds the inflammatory response contained predominantly macrophages whilst their morphological untreated variants showed mixed cell infiltrates rich in neutrophils. Predominant macrophages based response creates more favorable environment for healing. In FKDP-dressed wounds the number of microhemorrhages was also significantly decreased (p < 0.05) as compared with undressed wounds. Applied keratin dressing favors reconstruction of a more regular skin structure and assures better cosmetic effect in terms of scar formation and appearance. In conclusion, fur keratin-derived protein dressings significantly accelerated wound healing in the mouse model. Further studies are needed to determine the molecular mechanisms involved in the multilayer wound healing process and to assess the possible use of these dressings for medical purposes.

Different blood pressure responses to opioids in 3 rat hypertension models: role of the baseline status of sympathetic and renin-angiotensin systems.

Opioids interact with sympathetic and renin-angiotensin systems in control of mean arterial pressure (MAP). Our earlier finding that biphalin, a synthetic enkephalin analogue, decreased MAP in anaesthetized spontaneously hypertensive rats (SHR) prompted us to further explore this action, to get new insights into pathogenesis of various forms of hypertension. Biphalin effects were studied in SHR, uninephrectomized rats on a high-salt diet (HS/UNX), and rats with angiotensin-induced hypertension (Ang-iH). Besides MAP, renal and iliac blood flows (RBF, IBF) and vascular resistances were measured. In anaesthetized and conscious SHR, biphalin (300 µg·h-1·kg-1 i.v.) decreased MAP by ∼10 and ∼20 mm Hg, respectively (P < 0.001). In anaesthetized HS/UNX and normotensive rats, MAP increased by ∼6-7 mm Hg (P < 0.02); without anaesthesia, only transient decreases occurred. MAP never changed in Ang-iH rats. Morphine (1.5 mg·h-1·kg-1 i.v.) decreased MAP in HS/UNX but only transiently so without anaesthesia; such anaesthesia dependence of response was also seen in normotensive rats. Ang-iH rats never responded to morphine. Hypotensive effect in SHR only depends primarily on the reduction by biphalin of vascular responsiveness to increased sympathetic stimulation; such increase is well documented for SHR. No MAP response to biphalin or morphine in Ang-iH could depend on angiotensin-induced alterations of the vascular wall morphology and function.

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity.

New analogues of deltorphin I (DT I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 ), with the D-Ala residue in position 2 replaced by α-methyl-ß-azido(amino, 1-pyrrolidinyl, 1-piperidinyl or 4-morpholinyl)alanine, were synthesized by a combination of solid-phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to µ- and δ-opioid receptors. The affinity of analogues containing (R) or (S)-α-methyl-ß-azidoalanine in position 2 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. Peptide II, containing (S)-α-methyl-ß-azidoalanine in position 2, displayed excellent δ-receptor selectivity with its δ-receptor affinity being only three times lower than that of DT I.

Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

BACKGROUND: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. MATERIAL AND METHODS: Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. RESULTS: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. CONCLUSIONS: The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

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.

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

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.

Neuroprotective potential of biphalin, multireceptor opioid peptide, against excitotoxic injury in hippocampal organotypic culture.

UNLABELLED: Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. FINDINGS: (1) 0.025-0.1 µM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.

PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects.

BACKGROUND: The clinical treatment of various types of pain relies upon the use of opioid analgesics. However most of them produce, in addition to the analgesic effect, several side effects such as the development of dependence and addiction as well as sedation, dysphoria, and constipation. One solution to these problems are chimeric compounds in which the opioid pharmacophore is hybridized with another type of compound to incease antinociceptive effects. Neurotensin-induced antinociception is not mediated through the opioid system. Therefore, hybridizing neurotensin with opioid elements may result in a potent synergistic antinociceptor. RESULTS: Using the known structure-activity relationships of neurotensin we have synthesized a new chimeric opioid-neurotensin compound PK20 which is characterized by a very strong antinociceptive potency. The observation that the opioid antagonist naltrexone did not completely reverse the antinociceptive effect, indicates the partial involvement of the nonopioid component in PK20 in the produced analgesia. CONCLUSIONS: The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores overlap partially, expresses high antinociceptive tail-flick effects after central as well as peripheral applications.

Alcohol reverses depressive and pronociceptive effects of chronic stress in mice with enhanced activity of the opioid system.

The role of the opioid system in mediating effects of alcoholism and stress in depression is far from clear. We studied, therefore, the effects of chronic mild stress (CMS) and alcohol drinking on depression-like behavior and nociception in lines of mice selected for high (HA) or low (LA) swim stress-induced analgesia. Compared to the LA mice, the HA animals display up-regulation of opioid receptor system function and depression-like behavior in tail suspension test (TST). We report now that alcohol reverses depressive and pronociceptive effect of CMS in HA mice. In contrast, in LA mice CMS does not affect nociception or behavior in TST and the animals are not susceptible to alcohol under CMS. The results suggest that opioid system activity may determine the effects of alcohol on behavior under stress and, therefore, link predispositions to depression and to alcoholism.

Spinal cord hydrolysate ameliorate immunological reaction in experimental allergic encephalomyelitis.

The aim of this study was to use the hydrolysate of pig spinal cord proteins to induce oral tolerance in the animal model of sclerosis multiplex - experimental allergic encephalomyelitis. The female Lewis rats were fed with hydrolysate of pig spinal cord proteins in two doses for one week before immunization, which was induced by injection of guinea pig spinal cord homogenate. At the peak of clinical symptoms (the 13th day post immunization) the rats were sacrificed and the spleen removed. Splenocytes were suspended in a culture medium and placed in microculture plates. The cells were stimulated with homogenate. The cells were cultured for seven days. Proliferation of splenocytes was estimated by means of methyl-3H thymidine incorporation. In supernatants of cultures of splenocytes the level of cytokines INF-gamma, IL-10, IL-4, and TGF-gamma was measured. It was demonstrated that homogenate-induced splenocytes of hydrolysate-fed rats gave rise to low proliferation as compared to the controls used. The IFN-gamma was inhibited in hydrolysate-fed animals. The hydrolysate of pig spinal cord proteins has a modulatory effect on the immune reaction, particularly on the orally-induced antigen-specific modulation of autoimmune response.

Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers.

BACKGROUND: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1'-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20⁻100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2⁻20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug⁻target interactions.

Blood-brain barrier penetration by two dermorphin tetrapeptide analogues: role of lipophilicity vs structural flexibility.

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

Biphalin, a dimeric enkephalin analog, is under investigation as a potential, long-lasting medication of pain associated with chronic diseases, like cancer or AIDS. The role of cytokines, and splenocytes in anti-Friend leukemia virus (FLV) activity of biphalin, a synthetic opioid, and AZT was investigated in vitro. Mouse splenocytes inhibited FLV replication in Mus dunni (Dunni) cells when they were added to the cell culture. This inhibitory effect of splenocytes also was evident when cells were combined with biphalin and AZT as measured using a focus-forming assay. Under cell-free conditions, recombinant interferon gamma (IFNgamma), interleukin 2 (IL-2) and IL-4 directly inhibited the FLV reverse transcriptase (RT) activity by 27% to 36%. IFNgamma at 0.005 pg to 500 ng inhibited FLVRT activity by 61% to 80%. Acombination of 250 ng IFNgamma and 50 mug biphalin resulted in a 94% reduction of FLVRT activity, as compared with 61% inhibition by IFNgamma alone. The combination of AZT and IFNgamma, IL-2 or IL-4 also induced a stronger suppression of FLV RT activity than either cytokine or AZT used alone. In addition, cloned RT from Moloney murine leukemia virus (MMLV) was directly sensitive to inhibition by biphalin. Thus, the anti-FLV effects of splenocytes in combination with biphalin and AZT in cell culture are likely mediated to a large degree by the direct effect of cytokines. This antiviral activity of splenocytes or cytokines combined with chemotherapy, biphalin, and/or AZT, could be used as a complementary therapy to current approaches for retroviral infection and benefit acquired immunodeficiency syndrome (AIDS) patients. In conclusion, biphalin applied primarily as a new medicine for chronic pain treatment in AIDS patients may play a significant beneficial role as a component of antiviral HIV multidrug therapies.

Supranodose vagotomy eliminates respiratory depression evoked by dermorphin in anaesthetized rats.

The respiratory effects of stimulation of mu-opioid receptors were studied in spontaneously breathing anaesthetized rats that were either neurally intact or subjected to bilateral supranodosal vagotomy. An intravenous dermorphin bolus of 0.5 mg/kg evoked the apnea followed by breathing of reduced rate and compensatory augmentation of tidal volume, which resulted in an invariable minute ventilation. Cardiovascular effects consisted of hypotension and temporary fall in heart rate. In rats initially treated by supranodosal vagotomy, dermorphin did not evoke any respiratory and cardiovascular effects. These results indicate that vagal pathway and the nodose ganglia are involved in dermorphin-induced respiratory depression.

Cardiovascular and respiratory activity of PK20, opioid and neurotensin hybrid peptide in anesthetized and awake rats.

AIM: Chimeric compound - PK20 despite its therapeutic activity on nociceptive and inflammatory processes may affect respiration and blood pressure. Our objective was to evaluate influence of the hybrid composed of endomorphin-2 and neurotensin fragments on ventilation, heart rate and blood pressure in anesthetized and awake rats. METHODS: The effects of PK20 (1mg/kg) were studied either after its intravenous administration in anesthetized rats or intraperitoneal injection in awake state. Tidal volume and the timing components of the breathing pattern, arterial blood pressure, and heart rate were recorded. RESULTS: Intravenous administration of PK20 in the neurally intact rats evoked a dose-dependent apnoea followed by a transient insignificant increase in tidal volume and breathing rate. The blood pressure changes were biphasic: transient increase was replaced by prolonged hypotension. Midcervical vagotomy abrogated all post-PK20 respiratory effects. Hypotension was eliminated after blockade of neurotensin NTS1 receptor, while respiratory changes were reduced by blockade of both: NTS1 and µ opioid receptors. After PK20 intraperitoneal injection awake rats did not show any significant changes in ventilation and blood pressure. CONCLUSION: This chimeric peptide should be used with care via intravenous administration in anesthetized animals since PK20 may evoke respiratory apnoea and hypotension. Nevertheless, applied intraperitoneally in the same dose in conscious rats induced no adverse effects.

Hydrazone Linker as a Useful Tool for Preparing Chimeric Peptide/Nonpeptide Bifunctional Compounds.

The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type and length of linkers used to connect them are also crucial. In the present contribution, we describe compound 1 in which a typical opioid peptide sequence is combined with a fragment characteristic for neurokinin-1 receptor (NK1R) antagonists through a hydrazone bridge. The compound has a high affinity for µ- and δ-opioid receptors (IC50= 12.7 and 74.0 nM, respectively) and a weak affinity for the NK1R. Molecular modeling and structural considerations explain the observed activities. In in vivo test, intrathecal and intravenous administrations of 1 exhibited a strong analgesic effect, which indicates potential BBB penetration. This letter brings an exemplary application of the hydrazone linker for fast, facile, and successful preparation of chimeric compounds.

Arg-Ile-Tyr (RIY) derived from rapeseed protein decreases food intake and gastric emptying after oral administration in mice.

We previously reported that a bioactive tripeptide Arg-Ile-Tyr (RIY), which has been isolated as an inhibitor for angiotensin I-converting enzyme from the subtilisin digest of rapeseed protein, decreased blood pressure. In this study, we also found that RIY dose-dependently decreased food intake at a dose of 150 mg/kg after oral administration in fasted ddY male mice. The anorexigenic action of RIY was blocked by a cholecystokinin-1 CCK1 receptor antagonist, lorglumide. RIY also decreased the gastric emptying rate at a dose of 150 mg/kg and the RIY-induced delay of gastric emptying was blocked by lorglumide. However, RIY had no affinity for CCK1 receptor. Taken together, RIY decreased food intake and gastric emptying by stimulating CCK release.

An antihypertensive opioid: Biphalin, a synthetic non-addictive enkephalin analog decreases blood pressure in spontaneously hypertensive rats.

BACKGROUND: Endogenous opioid systems may be engaged in the control of arterial pressure (MAP), however, given the risk of addiction, opioid receptor agonists are not used in antihypertensive therapy. We examined cardiovascular effects of biphalin, a potentially non-addictive dimeric enkephalin analog, an agonist of opioid µ and δ receptors. METHODS: Biphalin was infused iv at 150µg/kg/h to anesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Along with MAP and heart rate (HR), renal blood flow (RBF) and iliac blood flow (IBF, a measure of hind limb perfusion) were measured using Transonic probes on renal and iliac artery, respectively. The effects of biphalin were compared with those of intravenous morphine (1.5mg/kg/h). RESULTS: In two SHR groups biphalin decreased MAP from 143±2 to 130±2 and from 177±4 to 167±3mmHg (p<0.001) while HR did not change or modestly decreased. The renal blood flow (RBF) increased modestly and both renal and hind limb vascular resistances decreased significantly (p<0.001). The responses were blocked by inhibition of peripheral opioid receptors with naloxone methiodide. Unlike in SHR, in WKY rats biphalin did not change MAP or vascular resistances. Morphine infusion decreased MAP in SHR from 169±6 to 150±6mmHg (less decrease in WKY) and significantly decreased RBF and IBF. CONCLUSION: Since biphalin, a non-addictive synthetic opioid, lowers MAP in SHR, a model of hypertension with pronounced neurogenic component, such analogs might find therapeutic application in human stress-induced hypertensive states. Biphalin's advantage is no associated reduction of renal perfusion.

An endomorphine analog ([d-Ala(2)]-Endomorphin 2, TAPP) lowers blood pressure and enhances tissue nitric oxide in anesthetized rats.

BACKGROUND: Activation of opioid receptors can alter cardiovascular function, an action possibly mediated by nitric oxide (NO). In this study we examined the effects of ([d-Ala(2)]-Endomorphin 2, TAPP), a synthetic opioid µ-receptor agonist, on blood pressure (MABP), tissue NO bioavailability and renal hemodynamics and excretion. METHODS: In acute experiments with anesthetized normotensive male Sprague-Dawley rats TAPP was given as a short iv infusion at a dose of 1.2 or 12mg/kg and then MABP, renal medullary NO signal (polarographic electrode), total renal blood flow (RBF, renal artery Transonic probe), renal regional perfusion (laser-Doppler fluxes) and renal excretion were simultaneously measured over 2h. RESULTS: After 1.2mg/kg dose MABP decreased progressively from 121±7 to 114±9mmHg (-6%, p<0.05) while kidney tissue NO signal increased from 29.1±2.7 to 31.7±3.1nA (6%, p<0.04). Both effects were prevented by Naloxone methiodide, a peripheral opioid receptor inhibitor. RBF and renal regional perfusion were not altered by either dose of TAPP; renal sodium excretion changes were highly variable and were not affected by Naloxone pretreatment. CONCLUSIONS: Briefly, we found that in anesthetized normotensive rats stimulation of peripheral opioid receptors with TAPP caused a prolonged decrease in arterial pressure, a change that was associated and probably causally related to an increase in tissue NO. The data suggest that synthetic opioids that do not penetrate the blood-brain barrier and are potentially non-addictive could be considered for antihypertensive therapy.