Coexistence of purino‐ and pyrimidinoceptors on activated rat microglial cells

Nucleotide‐induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100ngml −1 ) treated (non‐proliferating) rat microglial cells were recorded by the whole‐cell patch‐clamp technique. Most experiments were carried out on non‐proliferating microglial cells. ATP (100n m –1m m ), ADP (10n m –10m m ) and UTP (1μ m –100m m ), but not uridine (100μ m –10m m ) produced a slow outward current at a holding potential of 0mV. The effect of UTP (1m m ) did not depend on the presence of extracellular Mg 2+ (1m m ). The outward current response to UTP (1m m ) was similar in non‐proliferating and proliferating microglia. In non‐proliferating microglial cells, the ATP (10μ m )‐induced outward current was antagonized by suramin (300μ m ) or reactive blue 2 (50μ m ), whereas 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100μ m ) was inactive. By contrast, the current induced by UTP (1m m ) was increased by suramin (300μ m ) and was not altered by reactive blue 2 (50μ m ) or 8‐SPT (100μ m ). The current response to UTP (1m m ) disappeared when K + was replaced in the pipette solution by an equimolar concentration of Cs + (150m m ). However, the effect of UTP (1m m ) did not change when most Cl − was replaced with an equimolar concentration of gluconate − (145m m ). The application of 4‐aminopyridine (1m m ) or Cs + (1m m ) to the bath solution failed to alter the UTP (1m m )‐induced current. UTP (1m m ) had almost no effect in a nominally Ca 2+ ‐free bath medium, or in the presence of charybdotoxin (0.1μ m ); the inclusion of U‐73122 (5μ m ) or heparin (5mgml −1 ) into the pipette solution also blocked the responses to UTP (1m m ). By contrast, the effect of ATP (10μ m ) persisted under these conditions.I ‐V relations were determined by delivering fast voltage ramps before and during the application of UTP (1m m ). In the presence of extracellular Cs + (1m m ) and 4‐aminopyridine (1m m ) the UTP‐evoked current crossed the zero current level near−75mV. Omission of Ca 2+ from the Cs + (1m m )‐ and 4‐aminopyridine (1m m )‐containing bath medium or replacement of K + by Cs + (150m m ) in the pipette solution abolished the UTP current. Replacement of GTP (200μ m ) by GDP‐β‐S (200μ m ) in the pipette solution abolished the current evoked by UTP (1m m ). When the pipette solution contained Cs + (150m m ) instead of K + and in addition inositol 1,4,5,‐trisphosphate (InsP 3 ; 10μ m ), an inward current absolutely dependent on extracellular Ca 2+ was activated after the establishment of whole‐cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100mV, as measured by fast voltage ramps. A rise of the internal free Ca 2+ concentration from 0.01 to 0.5μ m on excised inside‐out membrane patches produced single channel activity with a reversal potential of 0mV in a symmetrical K + solution. The reversal potential was shifted to negative values, when the extracellular K + concentration was decreased from 144 to 32m m . By contrast, a decrease of the extracellular Cl − concentration from 164 to 38m m did not change the reversal potential. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP 3 . The depletion of the intracellular Ca 2+ pool appears to initiate a capacitative entry of Ca + from the extracellular space. This Ca 2+ then activates a Ca 2+ ‐dependent K + current.