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1.
Arterioscler Thromb Vasc Biol ; 36(1): 189-97, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26681756

RESUMO

OBJECTIVE: Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1-based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions. APPROACH AND RESULTS: PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01-2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 µmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥ 80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters. CONCLUSIONS: PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.


Assuntos
Plaquetas/efeitos dos fármacos , Peptídeos Penetradores de Células/administração & dosagem , Doença da Artéria Coronariana/terapia , Lipopeptídeos/administração & dosagem , Intervenção Coronária Percutânea , Inibidores da Agregação Plaquetária/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Receptor PAR-1/antagonistas & inibidores , Adulto , Idoso , Plaquetas/metabolismo , Peptídeos Penetradores de Células/efeitos adversos , Peptídeos Penetradores de Células/farmacocinética , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Lipopeptídeos/efeitos adversos , Lipopeptídeos/farmacocinética , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/efeitos adversos , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/farmacocinética , Testes de Função Plaquetária , Receptor PAR-1/metabolismo , Resultado do Tratamento
2.
Anal Biochem ; 468: 28-33, 2015 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-25232999

RESUMO

Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition.


Assuntos
Receptor de Endotelina B/imunologia , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Astrócitos/metabolismo , Western Blotting/métodos , Encéfalo/metabolismo , Células Cultivadas , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Ratos , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
3.
Pharmacol Res ; 64(3): 258-67, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21515378

RESUMO

Endothelin-1 (ET-1) is a pain mediator, elevated in skin after injury, which potentiates noxious thermal and mechanical stimuli (hyperalgesia) through the activation of ET(A) (and, perhaps, ET(B)) receptors on pain fibers. Part of the mechanism underlying this effect has recently been shown to involve potentiation of neuronal TRPV1 by PKCɛ. However, the early steps of this pathway, which are recapitulated in HEK 293 cells co-expressing TRPV1 and ET(A) receptors, remain unexplored. To clarify these steps, we investigated the pharmacological profile and signaling properties of native endothelin receptors in immortalized cell lines including HEK 293 and ND7 model sensory neurons. Previously we showed that in ND7/104, a dorsal root ganglia-derived cell line, ET-1 elicits a rise in intracellular calcium ([Ca(2+)](in)) which is blocked by BQ-123, an ET(A) receptor antagonist, but not by BQ-788, an ET(B) receptor antagonist, suggesting that ET(A) receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ET(A) and ET(B) receptors by RT-PCR and [(125)I]-ET-1 binding experiments confirms the slight predominance of ET(A) receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ET(B) receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca(2+)](in). Furthermore, reduction of ET(B) mRNA levels by siRNA does not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ET(B) receptor role in this response. However, in HEK 293 cells with low endogenous ET(A) mRNA levels, ET-1 does not induce a transient increase in [Ca(2+)](in). Observation of the [Ca(2+)](in) elevation in ND7/104 and HEK 293T cells in the absence of extracellular calcium suggests that ET-1 elicits a release of calcium from intracellular stores, and pretreatment of the cells with pertussis toxin or a selective inhibitor of phospholipase C (PLC) point to a mechanism involving Gαq/11 coupling. These results are consistent with the hypothesis that a certain threshold of ET(A) receptor expression is necessary to drive a transient [Ca(2+)](in) increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation.


Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , Endotelina-1/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor de Endotelina A/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Rim/citologia , Camundongos , Ratos , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Células Receptoras Sensoriais/metabolismo
4.
J Invest Dermatol ; 139(2): 412-421, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30287285

RESUMO

PAR2 has been proposed to contribute to lesion formation and intense itch in atopic dermatitis. Here, we tested the ability of a cell-penetrating pepducin, PZ-235, to mitigate the potentially deleterious effects of PAR2 in models of atopic dermatitis. PZ-235 significantly inhibited PAR2-mediated expression of inflammatory factors NF-κB, TSLP, TNF-α, and differentiation marker K10 by 94%-98% (P < 0.001) in human keratinocytes and suppressed IL-4 and IL-13 by 68%-83% (P < 0.05) in mast cells. In delayed pepducin treatment models of oxazolone- and DNFB-induced dermatitis, PZ-235 significantly attenuated skin thickening by 43%-100% (P < 0.01) and leukocyte crusting by 57% (P < 0.05), and it inhibited ex vivo chemotaxis of leukocytes toward PAR2 agonists. Daily PZ-235 treatment of filaggrin-deficient mice exposed to dust mite allergens for 8 weeks significantly suppressed total leukocyte and T-cell infiltration by 50%-68%; epidermal thickness by 60%-77%; and skin thickening, scaling, excoriation, and total lesion severity score by 46%-56%. PZ-235 significantly reduced itching caused by wasp venom peptide degranulation of mast cells in mice by 51% (P < 0.05), which was comparable to the protective effects conferred by PAR2 deficiency. Taken together, these results suggest that a PAR2 pepducin may confer broad therapeutic benefits as a disease-modifying treatment for atopic dermatitis and itch.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Dermatite Atópica/tratamento farmacológico , Prurido/tratamento farmacológico , Receptor PAR-2/antagonistas & inibidores , Animais , Peptídeos Penetradores de Células/uso terapêutico , Dermatite Atópica/etiologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Proteínas Filagrinas , Humanos , Queratinócitos , Masculino , Camundongos , Prurido/etiologia , Prurido/patologia , Receptor PAR-2/imunologia , Receptor PAR-2/metabolismo
6.
Pain ; 155(6): 1091-1101, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24569146

RESUMO

Endothelin (ET-1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre-exposure of ND7/104 immortalized sensory neurons to ET-1 (30nM) for 10min increased the proportion of cells responding to ATP (2µM) with an increase in intracellular calcium, an effect prevented by the ETA receptor-selective antagonist BQ-123. ET-1 (3nM) pre-exposure also increased the proportion of isolated mouse dorsal root ganglion neurons responding to ATP (0.2-0.4µM). Blocking ET-1-evoked increases in intracellular calcium with the IP3 receptor antagonist 2-APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET-1-mediated intracellular calcium increases. ET-1-sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using quantitative polymerase chain reaction and receptor-selective ligands in ND7/104 showed that ET-1-induced sensitization most likely involves the P2X4 receptor subtype. ET-1-sensitized calcium responses to ATP were strongly inhibited by broad-spectrum (TNP-ATP) and P2X4-selective (5-BDBD) antagonists, but not antagonists for other P2X subtypes. TNP-ATP and 5-BDBD also significantly inhibited ET-1-induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET-1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors.


Assuntos
Endotelina-1/toxicidade , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Receptores Purinérgicos P2X4/fisiologia , Células Receptoras Sensoriais/metabolismo , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo
7.
PLoS One ; 8(2): e56744, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23457608

RESUMO

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Ar , Conexinas/metabolismo , Queratinócitos/metabolismo , 1-Octanol/farmacologia , Trifosfato de Adenosina/deficiência , Carbenoxolona/farmacologia , Dor Crônica/metabolismo , Conexinas/antagonistas & inibidores , Células Epidérmicas , Homeostase/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Dermatopatias/metabolismo
8.
Pain ; 139(1): 90-105, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-18442883

RESUMO

Keratinocytes are implicated in sensory transduction and can influence nociception, but whether these contribute to chronic pain is not known. In neurons, voltage-gated sodium channels (Na(v)) are involved in neuropathic pain and are activated by depolarization. Since keratinocytes can also show changes in membrane potential, we used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the expression of sodium channels in these cells. Na(v)1.1, Na(v)1.6, and Na(v)1.8 were localized within keratinocytes in rat epidermis. In addition, sodium channels contribute to the release of ATP from rat keratinocytes in response to increased [K(+)](o), implicating sodium channels in keratinocyte ligand release and nociception. To examine whether keratinocytes may contribute to human pain states, we analyzed sodium channel expression in human skin biopsies from subjects with complex regional pain syndrome Type 1 (CRPS) and post-herpetic neuralgia (PHN) using immunohistochemistry. Control skin exhibited immunolabeling for Na(v)1.5, Na(v)1.6 and Na(v)1.7. In contrast, painful skin from CRPS and PHN subjects displayed Na(v)1.1, Na(v)1.2, and Na(v)1.8 immunolabeling, in addition to substantially increased signal for Na(v)1.5, Na(v)1.6, Na(v)1.7. These observations lead us to propose that pathological increases in keratinocyte sodium channel expression may contribute to pain by increasing epidermal ATP release, resulting in excessive activation of P2X receptors on primary sensory axons. Consistent with this hypothesis, animal models of neuropathic pain exhibit increases in subcutaneous ATP release and activity of primary sensory neurons, and peripheral administration of P2X antagonists has been shown to reduce neuropathic pain in humans.


Assuntos
Epiderme/fisiologia , Regulação da Expressão Gênica/fisiologia , Queratinócitos/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Dor/metabolismo , Canais de Sódio/biossíntese , Canais de Sódio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Epiderme/inervação , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Dor/genética , Ratos , Ratos Sprague-Dawley , Canais de Sódio/fisiologia
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