Homologous desensitization of bombesin-stimulated Ins(1,4,5)P3 production in Swiss 3T3 fibroblasts.

Desensitization is a process of adaptation by cells to a stimulus. It is characterised by a decrease in responsiveness of the cell and may be receptor-specific (homologous) or receptor-nonspecific (heterologous). Desensitization has been described for most receptor classes and for each of the second messenger systems [I-31. However, although agonist-induced desensitization has been observed in a variety of target cells, little is known of the molecular basis of desensitization of receptor-regulated phosphoinositidase C. Evidence exists for the modification of receptors [4,5], receptor down-regulation [6-81 and uncoupling of the receptor and the guanine nucleotide binding regulatory protein [9,10]. We have recently described mass measurements of Ins( 1,4,5)P3 and sn1.2 diacylglycerol in bombesin-stimulated Swiss 3T3 cells [ 1 I]. In the present study, we have used the Ins( 1,4,5)P3-specific mass assay [ 121 to demonstrate homologous desensitization of bombesin-stimulated Ins( 1,4,5)P3 production in this system, a phenomenon independent of protein kinase C. Swiss 3T3 cells were cultured in 24-well plates [ I I] and used for experiments when confluent and quiescent. Cells were washed and preincubated in Hank's buffered saline containing 2% (w/v) bovine serum albumin (fraction V) and 10 mM glucose (pH 7.4, HBG) prior to treatment with 100 pI buffer or bombesin as required. The medium was removed and the cells washed with buffer (2 x 600 11) prior to subsequent treatment with buffer or a second stimulation with bombesin. Incubations were terminated by the addition of 25 p1 icecold 10% (w/v) perchloric acid, the samples harvested and neutralised with 1.5 M KOW60 mM HEPES in the presence of Universal Indicator. Ins( 1,4,5)P3 mass was determined by competitive displacement of [3H]Ins( 1,4,5)P3 from bovine adrenal cortex microsomes [12]. Down-regulation of protein kinase C was by pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA, 400 nM) for 48 hr [ 131. Bombesin stimulation resulted in the transient formation of Ins (1,4,5)P3 (maximal response at 5-10 sec, 13.87 It 7.28 fold, n=4 experiments, returned to basal at 30 sec, data not shown). Bombesin pretreatment resulted in a loss of responsiveness of the cells to a subsequent stimulation with bombesin. For example: cells not pretreated, 5 sec control, 0.99 It 0.15, 5 sec bombesin, 11.03 f 2.5; cells pretreated for 90 sec with bombesin, 5 sec control, 1.52 f 0.18, 5 sec bombesin, 1.17 f 0.12, (n=3) from a single, typical experiment. The desensitization phenomenon was concentration-dependent (E.C.50 0.35 f 0.47 nM). Responsiveness was regained by incubation of bombeisn-pretreated cells in the absence of bombesin for a minimum of 10 min. However, resensitization was never complete within the timescale of the experiments (up to 60 min, data not shown). The lack of responsiveness was not due to depletion of the precursor lipid, PtdIns(4,5)Pz, which was not significantly diferent from unstimulated levels after 60 sec stimulation (data not shown). Down-regulation of protein kinase C by prolonged pretreatmmt with PMA has been shown to be complete in Swiss 3T3 cells [13]. A comparison was made between control (P-phorbol-pretreated) and protein kinase C-depleted (PMA-pretreated) cells. Homologous desensitization of the bombesin response was observed in both sets of cells (Fig. 1). Thus, the desensitization phenomenon was independent of protein kinase C activity. The site and mechanism of the desensitization observed remain unclear. Agonist-stimulation results in the activation of phosphoinositiadase C by a mechanism which involves a G-protein (Gp). Thus, potential sites at which desensitization might occur include the receptor, the receptor/Gp interaction, activation of phosphoinositidase C and phosphoinositidase C activity itself. That desensitization is occurring rather than just the activation of Ins( 1,4,5)P3 3-kinase and 5phosphatase is suggested by the lack of PtdIns(4,5)P2 hydrolysis in response to a second stimulation with bombesin (data not shown). In addition, the rate of formation of [3H]inositol phosphates is reduced (typically by 80%) after 1 min stimulation with bombesin in Swiss 3T3 cells labelled to isotopic equillibrium with [3H]inositol [ I l l . The mechanism of desensitization might involve modification of a protein or proteins, e.g. a conformational change in the receptor thereby reducing its affinity for bombesin or a change in the phosphorylation state of a regulatory protein. Homologous desensitization has been shown to be both dependent and independent of protein kinase C activity in other systems [ 14,151. However, the mechanism appears to be independent of protein kinase C activity which is surprising in view of the fact that PKC has been shown to negatively regulate phosphoinositidase C activity in these cells [16].