RESUMO
Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.
Assuntos
Conexinas/fisiologia , Junções Comunicantes/fisiologia , Animais , Comunicação Celular/fisiologia , Humanos , Imunidade Celular/fisiologia , Músculo Liso Vascular/fisiologia , Contração Miocárdica/fisiologia , RatosRESUMO
Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system
Assuntos
Humanos , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Imunidade Celular/fisiologia , Miocárdio/citologia , Comunicação Celular/fisiologia , Coração/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miocárdio/químicaRESUMO
Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.
Assuntos
Conexinas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Conexinas/classificação , Conexinas/genética , Primers do DNA/análise , DNA Complementar/análise , Endonucleases/análise , Humanos , Camundongos , Ratos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins
Assuntos
Animais , Humanos , Ratos , Camundongos , Conexinas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Conexinas/classificação , Conexinas/genética , Primers do DNA/análise , DNA Complementar/análise , Endonucleases/análise , Sensibilidade e Especificidade , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
The question arose as to whether the altered kinetic properties of the nicotinic acetylcholine receptor-ion channel complex (AChR) observed after muscle denervation could be due to post-translational modifications. Single AChR properties of in vitro denervated skeletal muscle were studied using the cell-attached patch-clamp technique. Patch-clamp recordings from junctional regions of fibers daily treated with cycloheximide, a protein synthesis inhibitor, and phenylmethylsulfonyl fluoride, a protease inhibitor, revealed single conductance class of acetylcholine (ACh)-activated currents after acute dissociation and several conductance classes 3-14 days after muscle denervation. alpha-Bungarotoxin blocked all conductance states induced by ACh. Biochemical assays carried out on the same experimental conditions disclosed that chronic treatment of the fibers with cycloheximide inhibited the incorporation of [3H]leucine into precipitable protein. The present findings provided evidence that the altered functional properties of nicotinic receptors after muscle denervation may occur without new transcription.