High glucose modulates P2X7 receptor-mediated function in human primary fibroblasts.
OBJECTIVE
Purinergic receptors are a family of newly characterized plasma membrane molecules involved in several and as yet only partially known cellular functions such as vascular reactivity, apoptosis and cytokine secretion. Little is known about the effect extracellular microenvironment has on their function. Fibroblasts share several features with smooth muscle cells and are an important constituent of the atherosclerotic plaque. Our aim was to evaluate the effect of high glucose concentration on ATP-mediated responses in human fibroblasts.


METHODS
Fibroblasts were obtained by skin biopsies and grown at two different glucose concentrations. We evaluated receptor expression by RT-PCR and immunoblotting and receptor localization by immunofluorescence. Plasma membrane potential and calcium changes were measured by fluorescent indicators. Apoptosis was determined by ethidium bromide staining and caspase-3 activation.


RESULTS
We show that cells grown in a medium with high glucose concentration underwent great ATP-mediated morphological changes, enhanced apoptosis, caspase 3 activation and interleukin-6 release. We identified P2X7 as the main purinergic receptor involved in these responses. Furthermore, high glucose concentration triggered the assembly of P2X7 into ring-like structures located at the periphery of the cells.


CONCLUSIONS
Given that ATP is frequently released into the extracellular milieu upon cell and tissue damage, secretory exocytosis or activation of plasma membrane transporters, we hypothesize that ATP receptors participate in the pathogenesis of vascular complications of diabetes.
Solini
A
A
Department of Clinical and Experimental Medicine, University of Ferrara, Italy.
Chiozzi
P
P
Falzoni
S
S
Morelli
A
A
Fellin
R
R
Di Virgilio
F
F
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability; D004789:Enzyme Activation; D004996:Ethidium; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D008564:Membrane Potentials; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling
C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D004996:Ethidium; D005947:Glucose; D002118:Calcium