[Morphologic evaluation and Ca2+ mobilization by glicose and acetylcholine in human pancreatic cells].
OBJECTIVE
The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method.


METHODS
The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization.


RESULTS
The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cells capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response.


CONCLUSIONS
The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.
Cardoso
Daniela Espinha
DE
Departamento de Biofísica, EPM, UNIFESP, São Paulo, SP. daniela@biofis.epm.br
França
Lucimar Pereira de
LP
Chinen
Elisângela
E
Moraes
Andréa A F Souza
AA
Ferreira
Alice Teixeira
AT
França
Jerônimo Pereira de
JP
D000109:Acetylcholine / Q000494:pharmacology*; D000704:Analysis of Variance; D020013:Calcium Signaling / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D053144:Cell Nucleus Shape; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000502:physiology; D007515:Islets of Langerhans / Q000737:chemistry / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D015388:Organelles / Q000737:chemistry; D043587:Receptor, Muscarinic M3 / Q000737:chemistry / Q000378:metabolism
D018679:Cholinergic Agonists; D007328:Insulin; D043587:Receptor, Muscarinic M3; D005947:Glucose; D000109:Acetylcholine