Bradykinin Transiently Activates Protein Kinase C in Swiss 3T3 Cells

The results presented here demonstrate that bradykinin, acting through a Bz subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-Z-loaded cells and by Wa’+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by oneand two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent M, = 80,000 (termed SOK), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of ‘251-epidermal growth factor binding or enhancement of CAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inosito1 phosphates in cells labeled with myo-[SH]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a IO-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.

Stimulation of many cell-surface receptors triggers the breakdown of phosphatidylinositol4,5bisphosphate by phospholipase C to produce two primary second messengers, di-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 (1,2). Diacylglycerol which is also generated from other sources (3), activates protein kinase C (2). Ins (1,4,5)P3 binds to a specific intracellular receptor (4) and releases calcium from intracellular stores (5,6) leading to an increase in [Ca"'];. Recent evidence, however, indicates that Ca2+ mobilization and activation of protein kinase C are not invariably linked. Receptor-mediated activation of protein kinase C can occur independently from inositol phospholipid turnover (7-g), and Ins(l,4,5)P3-mediated Ca2+ mobilization can take place without measurable activation of protein kinase C (10). Thus, the intensity, duration, and even the occurrence of protein kinase C activation must be determined directly rather than inferred from measurements of inositol phospholipid turnover. Bradykinin, a nonapeptide generated locally by proteolysis of a higher molecular weight precursor (ll), elicits biological responses in a wide variety of target cells including neuronal (12)(13)(14)(15), neuroendocrine (16,17), endothelial (18,19), epithelial (20,21), and fibroblastic (22)(23)(24) cells. The peptide initiates short term physiologic responses including pain and inflammation (11) and, in certain cells, long term mitogenesis (22,23,25). In all cellular systems, bradykinin rapidly increases the cellular level of Ins(1,4,5)P, and thereby causes Ca*+ mobilization from internal stores leading to a rise in [Ca2+li. In contrast, it has not been clearly demonstrated that bradykinin stimulates protein kinase C-mediated events in any of its target cells.
Bombesin and vasopressin also induce other protein kinase C-mediated events including transmodulation of epidermal growth factor (EGF) receptor affinity (34,43) and enhancement of CAMP accumulation (44,45). In the present study we used quiescent cultures of Swiss 3T3 cells to elucidate the early signals in the action of bradykinin.
As in other cell types, bradykinin caused a rapid  (Fig. lA, top). Bradykinin caused a transient increase in [Ca'+]i similar to those obtained with bombesin or vasopressin but distinct from that of platelet-derived growth factor, which induced Ca*+ mobilization after a 10-15-s delay (31). The B1 antagonist [des,Arg',Le$]bradykinin (54) did not prevent bradykinin-stimulated Ca2+ mobilization. In contrast, the B2 antagonist [D-Arg",Hyp3,Thi5.8,D-Phe7]bradykinin (55) caused a marked inhibition of Ca*+ mobilization induced by bradykinin but did not block the increase in [Ca2+li promoted by subsequent addition of bombesin (Fig.  lA). This suggests that bradykinin induces mitogenesis (22) and Ca2+ mobilization through a B, subtype receptor in Swiss 3T3 cells. Fig. 1B shows that addition of bradykinin to quiescent cultures of Swiss 3T3 cells labeled with 45Ca2' caused a marked increase in the rate of Ya2+ efflux, which was comparable to that elicited by bombesin or vasopressin. Bradykinin also stimulated cation efflux in Ca*+-free medium and reduced the cellular 'Wa*+ content by about 50% (data not shown). These results show that bradykinin, like bombesin and vasopressin, causes Ca*+ mobilization from internal stores in quiescent Swiss 3T3 cells. In agreement with this interpretation, addition of bradykinin to cells labeled with myo-[3H]inositol caused an increase from 450 + 10 cpm to 720 f 75 cpm (n = 3) in total inositol phosphates after 1 min of stimulation.

Bradykinin
Transiently Stimulates 8OK Phosphorylation-An increase in the phosphorylation of the acidic 80K cellular protein provides a specific marker of protein kinase C activation in intact Swiss 3T3 cells (34,37,39,40) and other cell types (41,42 of 80K phosphorylation induced by bradykinin after 10 min was greatly reduced compared with that stimulated by PDBu at 1 or 10 min or by bradykinin at 1 min (Fig. 2). The results presented in Fig. 2 prompted us to undertake a detailed kinetic analysis of 80K phosphorylation by bradykinin. Fig. 3A shows that 80K phosphorylation stimulated by 19 nM bradykinin reached peak values at 1 min of exposure (300% increase) and thereafter, rapidly declined to only 40% over the basal level. A similar kinetics was obtained when bradykinin was added at 40 nM (results not shown). In contrast to the transient stimulation of 80K phosphorylation in response to bradykinin, addition of either bombesin, vasopressin, or PDBu caused a sustained phosphorylation of 80K up to 10 min of incubation (Fig. 3B). Furthermore, the formation of diacylglycerol induced by bradykinin after 10 min was markedly reduced compared with that stimulated by bombesin or vasopressin at 1 or 10 min or by bradykinin at 1 min (Fig. 3A, inset). The possibility that bradykinin may activate either a diacylglycerol lipase or kinase, which would diminish the pool of diacylglycerol available to protein kinase C, or a protein phosphatase which could reverse the phosphorylation of 80K was considered.
No inhibitory effect on 80K phosphorylation was found either when bradykinin was added to the cells 10 min prior to the subsequent addition of bombesin, vasopressin, or PDBu or when bradykinin was added simultaneously with these agents (data not shown). In contrast, exposure of 3T3 cells to bradykinin for 10 min blocked the ability of this peptide to stimulate a second transient increase in 80K phosphorylation (  (Fig. 4A). We conclude that the transient increase in 80K phosphorylation induced by bradykinin reflects rapid but transient activation of protein kinase C.
As shown above (Fig. l), the mobilization of Ca2+ induced or the neuropeptides bombesin and vasopressin leads to a marked reduction in the affinity of the EGF receptor for EGF (34,43,59,60), a process called transmodulation. Fig. 5 demonstrates that bradykinin failed to cause transmodulation of the EGF receptor as shown at various concentrations of either bradykinin or Y-EGF and after different times of exposure to bradykinin.
In parallel cultures, bombesin which causes sustained activation of protein kinase C, as shown previously, decreased the binding of lz51-EGF in a time-and dose-dependent manner. Activation of protein kinase C by phorbol esters or neuropeptides is known to enhance the accumulation of CAMP in response to forskolin and other adenylate cyclase activators (44,45). As shown in Fig. 6, PDBu, vasopressin, and bombesin markedly enhanced (4-6-fold) the accumulation of CAMP in response to forskolin.
In contrast, bradykinin even at 1 pM caused only a slight enhancement of CAMP accumulation in parallel cultures. Thus, transient stimulation of protein kinase C by bradykinin is not sufficient to elicit either EGF receptor transmodulation or enhancement of CAMP accumulation. Effect of Bradykinin on the Accumulation of Inositol Phosphates-A plausible mechanism that accounts for the results described in this study is that unlike bombesin and vasopressin receptors, the occupied BS subtype bradykinin receptor   Cultures were washed and incubated at 37 "C in DMEM in the presence of 12.5 pM forskolin alone (cant) or with 1 pM bradykinin (Brk), 18 nM vasopressin (Vso), 200 nM PDBu (PDBu), or 6.2 nM bombesin (Born). After 1 h the cellular content of CAMP was measured. Data are mean f S.E., n = 9, obtained from three individual experiments. becomes rapidly uncoupled from phospholipase C stimulation. To examine this possibility, we performed a kinetic analysis of the effect of these peptides on the total accumulation of inositol phosphates in quiescent Swiss 3T3 cells labeled with myo-[3H]inositol and incubated in the presence of 20 mM LiCl. Fig. 7 shows that bombesin and vasopressin stimulated a linear accumulation of inositol phosphates over a period of 10 min. In contrast, bradykinin caused a rapid accumulation of inositol phosphates which reached a plateau after 2.5 min of incubation.
The attainment of a plateau in the presence of lithium strongly suggests that bradykinin stimulated a transient increase in the production of inositol phosphates. In the present study we demonstrate that bradykinin induced a transient increase in the phosphorylation of the acidic 80K protein, which has been identified as a major and specific substrate of protein kinase C in Swiss 3T3 cells and other cells and tissues (34)(35)(36)(37)(38)(39)(40)(41)(42). Proteins closely related to fibroblast 80K have been purified from bovine (63) and rat brain (51,64). The cDNA encoding the bovine protein-termed myristoylated alanine-rich C kinase substrate or MARCKS has been cloned and sequenced (65) and specific protein kinase C phosphorylation sites have been identified (66). Comparison of the amino acid sequences of proteolytic peptides generated from the rat 80K protein (67) with the deduced sequence of MARCKS (65) has suggested the existence of a family of protein kinase C substrates which does not exhibit significant homology to other known cellular proteins. Collectively, these findings indicate that an increase in the phosphorylation of 80K in intact cells provides a specific marker for protein kinase C activation.
Indeed, prolonged exposure of intact Swiss 3T3 cells to PDBu, a treatment known to down-regulate protein kinase C (56-58) blocked the transient increase in 80K phosphorylation by bradykinin. A salient feature of our results is that the kinetics of 80K phosphorylation in bradykinin-treated cells are sharply different from those observed in cells stimulated by other agents that activate protein kinase C including PDBu (39), exogenous diacylglycerols (36), platelet-derived growth factor (36), serum (68), as well as the mitogenic peptides bombesin (34) and vasopressin (37). Previous work from this laboratory has shown that removal of PDBu, bombesin, or vasopressin re-sulted in rapid reversal of 80K phosphorylation (69). These findings are specially relevant to the results presented here because they show that the 80K phosphoprotein is rapidly dephosphorylated by cellular protein phosphatases when the activation of protein kinase C is reversed. We conclude that the transient increase in 80K phosphorylation induced by bradykinin reflects transient and reversible activation of protein kinase C.
It is well established that bombesin and vasopressin, acting through their own distinct receptors (22,(70)(71)(72) stimulate sustained activation of protein kinase C which induces transmodulation of EGF receptor (34,43) and enhancement of CAMP accumulation (44,45). Interestingly, our results show that bradykinin neither reduced lz51-EGF binding nor enhanced CAMP accumulation.
In contrast, bombesin and vasopressin elicited these responses under identical experimental conditions. These findings support the conclusion that the rapid but transient activation of protein kinase C induced by bradykinin is not sufficient to evoke molecular responses through this signal-transduction pathway. This distinguishes signal-transduction through the BP subtype bradykinin receptor from all other agents that stimulate protein kinase C, particularly the peptides bombesin and vasopressin. To gain further insight into the differences in the early signaling events initiated by bradykinin, bombesin, and vasopressin, we examined the effects of these peptides on the formation of inositol phosphates and diacylglycerol as a function of time. Bradykinin caused a rapid but transient increase in the production of inositol phosphates and diacylglycerol. In contrast, both bombesin and vasopressin induced a sustained increase in the formation of these metabolites. These findings suggest that occupancy of Bz subtype bradykinin receptor by agonist is followed by rapid homologous receptor desensitization.
Indeed, exposure to bradykinin markedly and selectively attenuated the ability of this peptide to stimulate transient increase in 80K phosphorylation. We propose that bradykinin triggers phosphatidylinositol4,5-bisphosphate hydrolysis leading to Ins(l,4,5)P3-mediated Ca2+ mobilization but causes only a transient activation of protein kinase C due to rapid uncoupling of occupied bradykinin receptors from phospholipase C. Ligand-induced receptor phosphorylation has recently been shown to act as a mechanism to rapidly attenuate signal transduction through P-adrenergic receptors in a specific manner (73). Whether or not a similar mechanism is applicable to bradykinin must await further investigation. Regardless of the precise molecular mechanism, our findings show that the early signaling events elicited by bradykinin can be distinguished from those induced by bombesin and vasopressin. The results indicate an important mechanism by which ligands that stimulate phosphoinositide turnover and Ca*+ mobilization induce distinct patterns of molecular events in their target cells.