Fernando A. Gonzalez

Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization.

The stimulation of calcium efflux from Swiss 3T6 mouse fibroblasts by extracellular ATP was studied. It was found that the cells could be desensitized to ATP by a previous exposure to the nucleotide, lending support to the theory that this is a receptor mediated process. Another ATP-receptor mediated process in Swiss 3T6 cells, that is also subject to desensitization, causes the permeabilization of the plasma membrane to nucleotides and other normally impermeant compounds [Gonzalez et al., J. Cell. Physiol. 139:109 (1989)]. Here we demonstrate that selective desensitization of the ATP-dependent calcium mobilization pathway can be achieved without affecting ATP-induced permeabilization. Data are presented in support of the existence of multiple ATP-receptors (purinoceptors) in Swiss 3T6 cells.

The Cloning and Expression of G Protein-Coupled P2Y Nucleotide Receptors

The existence of cell surface receptors for extracellular adenine nucleotides in mammalian cells has been postulated for decades (Burn-stock, 1972), based on scores of reports on diverse responses to these molecules in a wide variety of tissues and cell types (Dubyak and El-Moatassim, 1993). Pharmacological studies have provided evidence for the expression of two major types of nucleotide receptors: those belonging to the G protein-coupled receptor family and those belonging to the ligand-gated ion channel family. Nucleotide receptors (P2 purinergic receptors) are distinct from P1 receptors for adenosine, and, from results of earlier studies, were thought to be selective for purine nucleotides, in particular adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and various analogs, including 2MeSATP, α,β-MeATP, and β,γ-MeATP. Recent studies have demonstrated that uridine nucleotides are equally or more effective than adenine nucleotides in activating several G protein-coupled P2 receptor subtypes, raising a question as to the appropriateness of referring to this family of receptors as “purinergic.” Nonetheless, nucleotide receptors have retained the designation “P2,” which now refers to both their purine- and/or pyrimidine-based nucleotide agonists.

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: Studies on individual cells using quantitative digital video fluorescence microscopy

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.