Pertussis toxin inhibits norepinephrine-stimulated inositol phosphate formation in primary brain cell cultures

Norepinephrine (NE) increased formation of [3H]inositol phosphates ( [3H]InsPs) in primary cultures of neuronal and glial cells from 1-day-old rat brain. This response appeared to be mediated by alpha 1-adrenergic receptors, because prazosin was 40-fold more potent than yohimbine in blocking it. Pretreatment with pertussis toxin (PTX) dose-dependently decreased this response by 70-80%. The IC50 for PTX (7 ng/ml) was similar to that for blocking of alpha 2-adrenergic receptor-mediated decreases in cyclic AMP accumulation in the same cells. PTX pretreatment caused only a small, not statistically significant, inhibition of the [3H]InsP response to the muscarinic cholinergic receptor agonist carbachol in these cells. Radioligand binding studies showed that both neuronal and glial cultures contained mixed populations of alpha 1a- and alpha 1b-adrenergic receptor subtypes. Selective inactivation of the alpha 1b population by chloroethylclonidine reduced NE-stimulated [3H]InsP formation by 25 +/- 6%. Pretreatment with both PTX and chloroethylclonidine caused additive decreases (90 +/- 3%) in the NE response. NE-stimulated [3H]InsP formation was partially dependent on extracellular calcium, because it was decreased 64 +/- 6% by removal of calcium and 56 +/- 13% by addition of 1 mM CdCl2, although it was not affected by 1 microM nifedipine. These results suggest that NE stimulates [3H]InsP formation in neuronal and glial cultures through a pertussis toxin-sensitive guanine nucleotide-binding protein. This response appears to be mediated primarily by the alpha 1a subtype and may be subsequent to calcium influx.