RESUMO
Live cell imaging of the movement of various membrane-bounded organelle cargos has enhanced our understanding of their function. Eukaryotic cells utilize microtubules and two classes of microtubule-based motor proteins, cytoplasmic dynein and members of the kinesin family, to deliver a variety of membrane-bounded organelles and other cargos to their appropriate locations. In order to better understand the functions and regulation of cytoplasmic dynein, we developed a method to study its location and motility in living cells. The technique takes advantage of the long thin axons of cultured hippocampal neurons. We use calcium phosphate to transfect fluorescent-tagged dynein intermediate chain (IC) subunits (DYNC1I) into cultured neurons. When the ICs are expressed at low levels, they are effective probes for the location of the cytoplasmic dynein complex in axons when living cells are imaged with fluorescence microscopy. The fluorescent subunit probes can be used to identify specific cargos of dynein complexes with different IC isoforms as well as the kinetic properties of cytoplasmic dynein.
Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Dineínas do Citoplasma/metabolismo , Quimografia/métodos , Animais , Fosfatos de Cálcio , Células Cultivadas , Embrião de Mamíferos/metabolismo , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Microtúbulos/metabolismo , Ratos , Transfecção , Proteína Vermelha FluorescenteRESUMO
Structure-activity studies were performed on the alpha(1A)-adrenoceptor (AR) selective agonist N-[5-(1H-imidazol-4-yl)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide (4). Compounds were evaluated for binding activity at the alpha(1A), alpha(1b), alpha(1d), alpha(2a), and alpha(2B) subtypes. Functional activity in tissues containing the alpha(1A) (rabbit urethra), alpha(1B) (rat spleen), alpha(1D) (rat aorta), and alpha(2A) (rat prostatic vas deferens) was also evaluated. A dog in vivo model simultaneously measuring intraurethral pressure (IUP) and mean arterial pressure (MAP) was used to assess the uroselectivity of the compounds. Many of the compounds that were highly selective in vitro for the alpha(1A)-AR subtype were also more uroselective in vivo for increasing IUP over MAP than the nonselective alpha(1)-agonists phenylpropanolamine (PPA) (1) and ST-1059 (2, the active metabolite of midodrine), supporting the hypothesis that greater alpha(1A) selectivity would reduce cardiovascular side effects. However, the data also support a prominent role of the alpha(1A)-AR subtype in the control of MAP.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Imidazóis/síntese química , Naftalenos/síntese química , Sulfonamidas/síntese química , Tetra-Hidronaftalenos/síntese química , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Cães , Feminino , Imidazóis/química , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Naftalenos/química , Naftalenos/farmacologia , Coelhos , Ensaio Radioligante , Ratos , Receptores Adrenérgicos alfa 1 , Baço/efeitos dos fármacos , Baço/fisiologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tetra-Hidronaftalenos/química , Tetra-Hidronaftalenos/farmacologia , Uretra/efeitos dos fármacos , Uretra/fisiologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
N-[3-(1H-Imidazol-4-ylmethyl)phenyl]ethanesulfonamide (ABT-866, 1) is a novel alpha(1) agent having the unique profile of alpha(1A) (rabbit urethra, EC(50) = 0.60 microM) agonism with alpha(1B) (rat spleen, pA(2) = 5.4) and alpha(1D) (rat aorta, pA(2) = 6.2) antagonism. An in vivo dog model showed 1 to be more selective for the urethra over the vasculature than A-61603 (2), ST-1059 (3, the active metabolite of midodrine), and phenylpropanolamine (4).