Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD₅₀) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD₅₀ of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.     

Comparative study between peripherally and centrally acting sublethal and lethal doses of Leiurus quinquestriatus scorpion venom in rabbits: The usefulness of the sodium channel blocker lidocaine

Background

Scorpion envenomation is common among desert dwellers, affecting several systems and resulting in multiple organ dysfunction (MOD) or failure (MOF), mainly due to their action on Na⁺ channels. Although scorpion venoms toxins do not pass the blood brain barrier, their CNS effects are prominent, occurring in conjunction with, or as an aftermath of peripheral actions of the venom.

Objective

To determine the ability of venom of the common scorpion Leiurus quinquestriatus (LQQ) to induce MOD or MOF when injected into rabbits in micro quantities centrally (intracerebroventricularly, i.c.v.) or macro amounts peripherally (s.c. or i.v.). Also, to assess if the Na⁺ channel blocker lidocaine can protect rabbits from the resultant manifestations.

Methods

Rabbits were injected with LQQ venom centrally or peripherally, in either sublethal or lethal doses, and MOD or MOF determined by assessing: cardiac output (CO), estimated hepatic blood flow (EHBF), biochemical parameters indicative of cardiac/hepatic/renal and pancreatic functions, blood pressure (BP), survival, lung/body index (LBI, indicative of pulmonary edema), and/or histological changes in hearts, lungs, livers plus kidneys. In pre-treatment experiments, lidocaine was injected 40 min before venom and protective ability examined.

Results

LQQ venom in sublethal doses caused comparable significant reductions (vs control) in CO and EHBF when injected i.c.v. (2 μg kg⁻¹) or s.c. (0.2 mg kg⁻¹). Both routes caused gradual dose-related enhanced levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatinine, glucose and amylase, indicating MOD. Also, characteristic venom-induced changes in BP were evident after lethal doses of venom i.v. (0.5 mg kg⁻¹) or i.c.v. (3 μg kg⁻¹). Histological changes in the organs plus LBI were comparable after i.c.v. and i.v. venom injection, with animals ultimately exhibiting MOF. Lidocaine (1 mg kg⁻¹ i.v., then infusion 50 μg kg⁻¹ min⁻¹, 30 min before venom), protected the animals from MOF evoked by lethal doses of the venom (whether injected centrally or peripherally), as evidenced by the amelioration of the venom’s effects on blood pressure, LBI, survival and multiple organ histopathological manifestations.

Conclusion

LQQ venom, whether injected centrally or peripherally caused comparable systemic dose-dependent MOD or MOF, with the latter attenuated by the Na⁺ channel blocker lidocaine, indicating a role for Na⁺ channels.     

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with K_D values below pM (k_off < 1 × 10⁻⁷ s⁻¹) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.     

Effect of Different N:P Ratios on the Growth,Toxicity, and Toxin Profile of Gymnodinium catenatum (Dinophyceae) Strains from the Gulf of California

The harmful microalgae Gymnodinium catenatum is a unique naked dinoflagellate that produces paralytic shellfish poisoning toxins (PSTs). This species is common along the coasts of the Mexican Pacific and is responsible for paralytic shellfish poisoning, which has resulted in notable financial losses in both fisheries and aquaculture. In the Gulf of California, G. catenatum has been related to mass mortality events in fish, shrimp, seabirds, and marine mammals. In this study, the growth, toxin profiles, and toxin content of four G. catenatum strains isolated from Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were evaluated with different N:P ratios, keeping the phosphorus concentration constant. All strains were cultivated in semi-continuous cultures (200 mL, 21.0 °C, 120 µmol photon m⁻²s⁻¹, and a 12:12 h light-dark cycle) with f/2 + Se medium using N:P ratios of: 4:1, 8:1, 16:1, 32:1, and 64:1. Paralytic toxins were analyzed by HPLC with fluorescence detection. Maximum cellular abundance and growth were obtained at an N:P ratio of 64:1 (3188 cells mL⁻¹ and 0.34 div day⁻¹) with the BAMAZ and BAPAZ strains. A total of ten saxitoxin analogs dominated by N-sulfocarbamoyl (60–90 mol%), decarbamoyl (10–20 mol%), and carbamoyl (5–10 mol%) toxins were detected. The different N:P ratios did not cause significant changes in the PST content or toxin profiles of the strains from both bays, although they did affect cell abundance.     

In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.     

Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities

Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10⁻¹⁰ M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10⁻¹⁰ M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.     

In Vitro Toxic Effects of Puff Adder (Bitis arietans) Venom,and Their Neutralization by Antivenom

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3–5, unpaired t-test). Addition of antivenom at t₅₀ failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.     

Long-term antibiotic use during early life and risks to mental traits: an observational study and gene–environment-wide interaction study in UK Biobank cohort

The relationships between long-term antibiotic use during early life and mental traits remain elusive now. A total of 158,444 subjects from UK Biobank were used in this study. Linear regression analyses were first conducted to assess the correlations between long-term antibiotic use during early life and mental traits. Gene–environment-wide interaction study (GEWIS) was then performed by PLINK2.0 to detect the interaction effects between long-term antibiotic use during early life and genes on the risks of mental traits. Finally, DAVID tool was used to conduct gene ontology (GO) analysis of the identified genes interacting with long-term antibiotic use during early life. We found negative associations of long-term antibiotic use during early life with remembrance (p value=1.74 × 10⁻⁶, b = −0.10) and intelligence (p value=2.64 × 10⁻²⁶, b = −0.13), and positive associations of long-term antibiotic use during early life with anxiety (p value = 2.75 × 10⁻⁴⁷, b = 0.12) and depression (p value=2.01 × 10⁻¹⁹⁵, b = 0.25). GEWIS identified multiple significant genes-long-term antibiotic use during early life interaction effects, such as ANK3 (rs773585997, p value = 1.78 × 10⁻⁸) for anxiety and STRN (rs140049205, p value = 1.88 × 10⁻⁸) for depression. GO enrichment analysis detected six GO terms enriched in the identified genes interacting with long-term antibiotic use during early life for anxiety, such as GO:0030425~dendrite (p value = 3.41 × 10⁻²) and GO:0005886~plasma membrane (p value = 3.64 × 10⁻³). Our study results suggest the impact of long-term antibiotic use during early life on the development of mental traits.Subject terms: Anxiety, Depression     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

Simultaneous Effect of Temperature and Irradiance on Growth and Okadaic Acid Production from the Marine Dinoflagellate Prorocentrum belizeanum

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I₀). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·m⁻²·s⁻¹), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ_m) was 0.204 day⁻¹ at 25 °C and 40 µE·m⁻²·s⁻¹. Photo-inhibition was observed at I₀ > 40 μEm⁻²s⁻¹, leading to culture death at 120 µE·m⁻²·s⁻¹ and 28 °C. Cells at I₀ ≥ 80 µE·m⁻²·s⁻¹ were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ_m-I₀ curves and another empirical model for relating µ_m-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I₀-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.     

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Background and purpose:

The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis.

Experimental approach:

BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund''s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 µg, 5 µg, 10 µg, 20 µg or 40 µg·50 µL⁻¹). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines.

Key results:

Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 µg·50 µL⁻¹) was more effective than ‘free’ P10 emulsified in Freund''s adjuvant (20 µg·50 µL⁻¹), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 µg·50 µL⁻¹) were most effective. Treatment with P10 emulsified in Freund''s adjuvant (20 µg·50 µL⁻¹) or P10 entrapped within PLGA (1 µg·50 µL⁻¹) were accompanied by high levels of interferon-gamma in lung.

Conclusions and implications:

Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.     

Effects of moexiprilat on oestrogen-stimulated cardiac fibroblast growth

1. The effects of 2-2-(1-(ethoxycarbonyl)-3-phenylpropyl)-[amino-oxopropyl]−6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (moexiprilat), 17β-oestradiol (E₂), oestrone (ES) and angiotensin II (AII) on growth and activation of oestrogen receptors and the immediate-early gene egr-1 were investigated in neonatal rat cardiac fibroblasts of female and male origin.
2. In BrdU proliferation assays, oestrone (10⁻⁷–10⁻⁹ M) stimulated cardiac fibroblast growth in a concentration-dependent fashion (maximum at 10⁻⁷ M, 4.0 fold±0.14 in female and 3.1 fold±0.06 in male cells, n=9, P<0.05), while E₂ (10⁻⁷–10⁻⁹ M) had no effect. Moexiprilat (10⁻⁷ M) completely inhibited oestrone-induced cardiac fibroblast growth.
3. Angiotensin II (10⁻⁷ M) induced cardiac fibroblast growth (female 4.1 fold±0.1/male 3.9 fold±0.2; n=9, P<0.05). Angiotensin II induced oestrogen receptor (maximum 21.8 fold at 60 min) and egr-1 (maximum 47.5 fold at 60 min) expression in a time-dependent fashion.
4. In immunoblot experiments, oestrogen activated oestrogen receptor (ES: 12.8 fold±2.0; E₂: 14.7 fold±4.9; n=3, P<0.05) and egr-1 (ES: 5.1 fold,±0.24; E₂: 3.8 fold,±0.25; n=3, P<0.05) expression. The induction of oestrogen receptor and egr-1 protein expression was time-dependent and inhibited by moexiprilat.
5. Our results show that oestrone and 17β-oestradiol reveal a significant difference in their potential to activate cardiac fibroblast growth in female and male cells and that oestrone-stimulated growth is inhibited by moexiprilat. The inhibition of oestrone-stimulated cardiac fibroblast growth by moexiprilat may contribute to the beneficial effects seen in postmenopausal women with hypertensive heart disease treated with ACE inhibitors.

     

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity,Toxin Profile,and Co-Occurrence with Cyclic Imines

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC–MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg⁻¹ for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg⁻¹ for the LC–MS/MS method, with a good correlation between both methods (r² = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious,hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3–4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 μg kg⁻¹ h⁻¹, i.v.)
2. The early (1–2 h) hypotension in SD rats (−11±3 mmHg; n=7) was significantly less than that in TG rats (−35±3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108±3 mmHg; 24 h LPS, 112±4 mmHg; TG rats: baseline, 171±2 mmHg; 24 h LPS, 169±3 mmHg). At this stage in the SD rats there was a renal vasodilatation (Δ vascular conductance, 29±10 [kHz mmHg⁻¹]10³) but not in TG rats (Δ vascular conductance 2±3[kHz mmHg⁻¹]10³).
3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 μg kg⁻¹ bolus, 600 μg kg⁻¹ h⁻¹) between 24 and 31 h in SD rats caused a fall in MAP of 16±2 mmHg accompanied by hindquarters vasodilatation (Δ vascular conductance 11±3 (kHz mmHg⁻¹)10³). In TG rats, under the same conditions, the fall in MAP was −60±6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (Δ vascular conductance, 23±5, 32±7, and 14±4 (kHz mmHg⁻¹)10³, respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats.
4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was −17±4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5±3, −4±5, and 12±4 (kHz mmHg⁻¹)10³, respectively.
5. Administration of the angiotensin (AT₁)-receptor antagonist, losartan (10 mg kg⁻¹, i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (−12±3 and −14±4 mmHg, respectively).
6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.

     

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor

Background and purpose:

Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs.

Experimental approach:

We studied the interactions of mamba venom fractions with α₁-adrenoceptors in binding experiments with ³H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle.

Key results:

AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K_i= 0.35 nM) and high specificity for the human α_1A-adrenoceptor subtype. We showed high selectivity and affinity (K_d= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k_on= 6 × 10⁶·M⁻¹·min⁻¹) with an unusually stable α_1A-adrenoceptor/AdTx1 complex (t_1/2diss= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine''s actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM.

Conclusions and implications:

AdTx1 is the most specific and selective peptide inhibitor for the α_1A-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.