RESUMO
This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4(+)CD25(-) effector and CD4(+)CD25(+)CD127(low) regulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis.
Assuntos
Linfócitos T CD4-Positivos/citologia , Regulação da Expressão Gênica , Psoríase/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Psoríase/imunologia , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser(79) and Raptor at Ser(792), was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α(1) and α(2) catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.
Assuntos
Proteínas Quinases Ativadas por AMP/biossíntese , Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Neoplasias Pancreáticas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Técnicas de Silenciamento de Genes , Glucose/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Serina-Treonina Quinases TORRESUMO
The psoriatic skin resembles wound healing, and it shows abnormalities at the basement membrane (BM), also in the non-lesional skin. Fibroblast-derived dermal periostin has well-known functions in wound healing and Th2-mediated diseases, such as atopic dermatitis. Here we show that serum periostin level was elevated in psoriatic patients, remarkably in the systemically treated ones. Obvious periostin positivity was detected in basal keratinocytes of the non-lesional, lesional, and previously-lesional psoriatic vs. healthy skin. Ex vivo skin models were generated to examine how different skin injuries affect periostin expression during wound healing. Our newly developed cultured salt-split model demonstrated that BM-injury induced periostin expression in basal keratinocytes, and periostin levels in the supernatant were also increased upon healing. In wound healing models, ß1-integrin expression was similarly induced. ß1-integrin blocking caused reduced periostin expression in in vitro scratch assay, indicating that ß1-integrin can mediate periostin production. In contrast to atopic dermatitis, psoriatic basal keratinocytes are in an activated state and show a stable wound healing-like phenotype with the overexpression of periostin. This abnormal BM-induced wound healing as a potential compensatory mechanism can be initiated already in the non-lesional skin present in the lesion and keratinocytes can remain activated in the healed skin.
Assuntos
Dermatite Atópica , Psoríase , Humanos , Dermatite Atópica/patologia , Queratinócitos/metabolismo , Pele/metabolismo , Psoríase/patologia , Cicatrização/genética , Membrana Basal/metabolismo , Integrinas/metabolismoRESUMO
We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.
Assuntos
DNA/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Animais , Biocatálise , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Cinética , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C/isolamento & purificação , Proteína Quinase D2 , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptores Acoplados a Proteínas G/agonistasRESUMO
Ramsay Hunt syndrome is a special form of herpes zoster which is typically characterized by peripheral facial palsy and unilateral herpetic vesicles on the ear. These symptoms are often accompanied by vestibulocochlear dysfunction and other neurological and ophthalmological symptoms. The diagnosis and therapy requires a multidisciplinary approach. The authors present a typical case where the early administration of combined antiviral and systemic corticosteroid therapy led to complete recovery. The authors emphasize the importance of early diagnosis and adequate combination therapy, which improves the prognosis of this disease.
Assuntos
Corticosteroides/uso terapêutico , Antivirais/uso terapêutico , Paralisia Facial/virologia , Herpes Zoster da Orelha Externa/diagnóstico , Herpes Zoster da Orelha Externa/tratamento farmacológico , Hospedeiro Imunocomprometido , Aciclovir/uso terapêutico , Corticosteroides/administração & dosagem , Adulto , Antivirais/administração & dosagem , Quimioterapia Combinada , Diagnóstico Precoce , Feminino , Herpes Zoster da Orelha Externa/complicações , Humanos , Comunicação Interdisciplinar , Resultado do TratamentoRESUMO
Photodynamic therapy involves - in dermatological practice usually exogenous - application of a photosensitizer then activation of accumulated protoporphyrin IX by light with an appropriate wavelength after a short incubation period. It is an evidence based method to treat certain non-melanoma skin cancers. During treatment when the excited protoporphyrin IX returns to base state, reactive oxygen species are formed leading to cell death in rapidly proliferating cells. Fluorescence of excited protoporphyrin IX can be used in diagnostics as well. In ultraviolet light, the photodamaged or neoplastic areas show coral red fluorescence which can clearly be distinguished from the much lower fluorescence of adjacent normal tissue. This process is suitable for exact determination of tumor margins so it can be used for planning surgical procedures or after photodynamic therapy at a follow up visit for the visualization of the therapeutic result. The present article reviews the literature of photodynamic diagnosis that is also used by the authors.
Assuntos
Carcinoma Basocelular/diagnóstico , Carcinoma Basoescamoso/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Fluorescência , Cirurgia de Mohs/métodos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Protoporfirinas/administração & dosagem , Neoplasias Cutâneas/diagnóstico , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/análogos & derivados , Apoptose , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basoescamoso/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células , Temperatura Baixa , Humanos , Dor/etiologia , Dor/prevenção & controle , Fotoquimioterapia/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [(3)H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G(1)/G(o) to S phase.
Assuntos
Azepinas/farmacologia , Benzofuranos/farmacologia , Bombesina/farmacologia , Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Células 3T3 , Animais , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Camundongos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/metabolismo , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
INTRODUCTION: The dry and scaly skin of psoriatic patients decreases the efficacy of ultraviolet B (UVB) phototherapy. Different agents are used to facilitate the transmission of light, but most of these preparations are cosmetically unfavorable. We have tested a novel preparation containing sodium hyaluronate and nicotinic acid (UV Fotogel®; Pernix Ltd.) with the double aim to improve the efficacy of UVB phototherapy and assess the cosmetic acceptability of the preparation. METHODS: Ninety patients with plaque psoriasis were enrolled in the study, of whom 44 received narrow-band UVB (NB-UVB) phototherapy. Prior to phototherapy, one side of the patient's body was treated with UV Fotogel while the other side served as a control. The other 46 patients used the preparation at their homes before regular sunbathing. The Local Psoriasis Severity Index (L-PSI), cosmetic acceptability and tolerability were recorded. The median values with the 25th and 75th percentiles (25p and 75p, respectively) were determined for the UV Fotogel-treated and control sites and then compared. RESULTS: The sides of the body to which UV Fotogel was applied prior to NB-UVB phototherapy had a significantly lower median L-PSI score than the non-treated control sides at the end of the treatment (1.0 [25p-75p: 0.0-2.0] vs. 2.0 [1.0-3.0], respectively). The application of UV Fotogel prior to sunbathing also led to a significant decrease in L-PSI score. There was a significant reduction in the median L-PSI score of patients at the final visit compared to baseline (2.5 [25p-75p: 1.5-3.5] vs. 6.0 [6.0-7.0], respectively). Use of the preparation was not accompanied by considerable adverse effects, and the patients found it cosmetically acceptable. Application of UV Fotogel prior to sunbathing was well tolerated by the patients, and the cosmetic acceptability was also good. CONCLUSION: UV Fotogel is potentially a useful device for enhancement of the efficacy of phototherapy in patients with psoriasis.
RESUMO
Non-melanoma skin cancers are the most common skin tumors. Because of their frequent localization on the face and hand, aesthetic aspects of the therapeutic procedures should also be considered. Surgical excision still remains the first choice, but recently several new alternative therapies have emerged, especially for the treatment of superficial skin cancer. Photodynamic therapy has become a widely accepted therapeutic method for certain non-melanoma skin tumors. Photodynamic therapy involves the use of light to activate a photosensitizer, localized in diseased tissues. Photosensitizers are tumor-selective: their accumulation in rapidly proliferating cells and newly formed blood vessels is significantly higher than in the surrounding healthy tissues. During photodynamic therapy, cytotoxic reactive oxygen species are formed from the photosensitizer, leading to changes in subcellular pathways or apoptosis of the cells. Efficacy of the photodynamic therapy has been proven in solar keratosis, superficial basal cell carcinoma and morbus Bowen, with significantly better cosmetic outcome than that of the conventional therapeutic methods. Side effects, like erythema, crusting, serous discharge, or oedema, are usually moderate, and dissolve rapidly. The present article summarizes the authors' experiences with photodynamic treatment (212 non-melanoma skin cancer patients were treated with PDT between December 2003 and January 2006), at the Department of Dermatology and Allergology, University of Szeged, Hungary, and reviews the literature of photodynamic therapy in dermatooncology.
Assuntos
Fotoquimioterapia , Neoplasias Cutâneas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Medicina Baseada em Evidências , Humanos , Hungria , Fotoquimioterapia/efeitos adversos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Antidrug antibodies have been shown to be associated with a loss of response during biologic therapy. Despite the potential association, there has been no report on the simultaneous monitoring of the following parameters in psoriasis: presence of neutralizing antibodies, plasma tumor necrosis factor (TNF)-α concentration, TNFi concentration and disease activity. Plasma concentrations of adalimumab, infliximab, etanercept and their respective antidrug antibodies, as well as plasma concentrations of TNF-α were measured in 77 psoriasis patients receiving biologic therapy, and the values were correlated with the clinical activity of the skin disease. Antidrug antibodies were identified in the plasma of 25% of infliximab-treated patients and 29.6% of adalimumab-treated patients, but not in the etanercept group. Clinical severity scores were significantly higher in the antibody-positive patients. In patients receiving infliximab or adalimumab therapy, the presence of antidrug antibodies was directly associated with reduced plasma TNF-inhibitor concentration and elevated plasma TNF-α level.
Assuntos
Adalimumab/imunologia , Anticorpos Neutralizantes/sangue , Fármacos Dermatológicos/imunologia , Etanercepte/imunologia , Infliximab/imunologia , Psoríase/tratamento farmacológico , Fator de Necrose Tumoral alfa/sangue , Adalimumab/sangue , Adalimumab/uso terapêutico , Adulto , Idoso , Terapia Biológica/métodos , Estudos Transversais , Fármacos Dermatológicos/sangue , Fármacos Dermatológicos/uso terapêutico , Etanercepte/sangue , Etanercepte/uso terapêutico , Feminino , Humanos , Infliximab/sangue , Infliximab/uso terapêutico , Masculino , Pessoa de Meia-Idade , Psoríase/imunologia , Índice de Gravidade de Doença , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto JovemRESUMO
AIM: To assess tumor necrosis factor-α (TNF-α), infliximab (IFX) concentrations, and antibodies against IFX molecules in patients with inflammatory bowel disease (IBD) who develop loss of response, side effects, or allergic reaction during anti TNF-α therapy. METHODS: Blood samples of 36 patients with response loss, side effects, or hypersensitivity to IFX therapy (Groupâ I) and 31 patients in complete clinical remission (Group II) selected as a control group were collected to measure trough serum TNF-α level, IFX, and anti-IFX antibody (ATI) concentration. We examined the correlation between loss of response, the development of side effects or hypersensitivity, and serum TNF-α, IFX trough levels, and ATI concentrations. RESULTS: The serum TNF-α level was shown to be correlated with the presence of ATI; ATI positivity was significantly correlated with low trough levels of IFX. ATIs were detected in 25% of IBD patients with loss of response, side effects, or hypersensitivity, however no association was revealed between these patients and antibody positivity or lower serum IFX levels. Previous use of IFX correlated with the development of ATI, although concomitant immunosuppression did not have any impact on them. CONCLUSION: On the basis of the present study, we suggest that the simultaneous measurement of serum TNF-α level, serum anti TNF-α concentration, and antibodies against anti TNF-α may further help to optimize the therapy in critical situations.
Assuntos
Anti-Inflamatórios/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Anticorpos/sangue , Hipersensibilidade a Drogas/etiologia , Monitoramento de Medicamentos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Fator de Necrose Tumoral alfa/sangue , Adolescente , Adulto , Idoso , Anti-Inflamatórios/sangue , Anti-Inflamatórios/imunologia , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/imunologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/imunologia , Infliximab , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Indução de Remissão , Fatores de Risco , Falha de Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto JovemRESUMO
Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3-10 nm) or with vasopressin, a different G(q)-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser(744) and Ser(748). Transphosphorylation targeted Ser(744), whereas autophosphorylation was the predominant mechanism for Ser(748) in cells stimulated with G(q)-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G(q)-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Mitose/fisiologia , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bombesina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Maleimidas/farmacologia , Camundongos , Mitose/efeitos dos fármacos , Neurotransmissores/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Quinase C/antagonistas & inibidores , Células Swiss 3T3 , Fatores de Tempo , Vasoconstritores/farmacologia , Vasopressinas/farmacologiaRESUMO
In previous studies, we found that apical and basolateral EGF receptors (EGFR) on primary canine gastric monolayers decreased paracellular permeability, evident by increased transepithelial electrical resistance (TER) and decreased flux of [(3)H]mannitol (MF). After studying monolayers in Ussing chambers, we now report that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H(+), evident by a slower decay in TER and an attenuated rise in MF. Enhanced tolerance to apical acid was evident within 10 min of treatment with apical EGF. Immunoneutralization of endogenous transforming growth factor (TGF)-alpha accelerated the drop in TER and the rise in MF in response to apical acidification; apical EGF reversed these effects. Study of monolayers cultured in Transwell inserts showed that immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We conclude that apical EGFR regulates the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a less apparent effect on the barrier to acid, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate a role for an apical EGFR ligand, which may be EGF or another member of the EGF family.
Assuntos
Receptores ErbB/fisiologia , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Fator de Crescimento Transformador alfa/fisiologia , Ácidos/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cães , Impedância Elétrica , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/imunologia , Mucosa Gástrica/citologia , Mucosa Gástrica/fisiologia , Soros Imunes/farmacologia , Membranas Intracelulares/metabolismo , Permeabilidade/efeitos dos fármacos , Fator de Crescimento Transformador alfa/imunologiaRESUMO
Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr(416) was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 microM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr(416) in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this G(s)-coupled receptor in primary epithelial cells.