Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets.

Histamine is known to be a mediator of inflammation. In order to understand the role of histamine in platelets, we have examined the effects of histamine on arachidonic acid (AA) release, cAMP accumulation, inositol trisphosphate production, and serotonin secretion. Incubation of rabbit (and human) platelets with histamine resulted in rapid increase of [3H]AA release from the platelets prelabeled with [3H]AA. The effect of histamine was blocked by the addition of H1 receptor antagonist mepyramine. Histamine did not substantially affect the cAMP content and inositol trisphosphate production. Histamine-stimulated AA release was not observed in digitonin-permeabilized platelets, whereas histamine acted synergistically with GTP or GTP analog, guanosine 5'-(3-O-thio)triphosphate. Histamine-stimulated, and GTP analog-dependent AA release was inhibited by guanosine 5'-(2-O-thio) diphosphate. The effects of three receptor stimulants, thrombin, norepinephrine, and histamine were both diminished by 1 microgram/ml of pertussis toxin treatment and by the antiserum against GTP-binding proteins (G proteins) treatment. However, the antiserum against beta gamma subunits of G proteins inhibited the histamine effect, not thrombin effect. 4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment enhanced histamine-stimulated AA release and serotonin secretion but inhibited thrombin-stimulated reactions. The effect of PMA was dose dependent and was due to enhance the coupling of histamine receptors and G proteins. The results show the existence of H1 histamine receptors which couple phospholipase A2 activation via pertussis toxin-sensitive G proteins. Histamine actions differ in sensitivities to anti-beta gamma antiserum treatment and PMA treatment from thrombin actions.

There are many lines of evidence suggesting that histamine might act as neurotransmitter in the central nervous system. Additionally, histamine is established to be a chemical mediator of allergies and inflammation (Schwartz et al., 1986a, 198613). Histamine action is believed to be mediated via cell surface receptors, which were classified to three distinct subtypes, Hi, Hz, and Ha-receptors, by pharmacological studies. The histamine actions produced by H1 receptors including bronchoconstriction, vasoconstriction, increased capillary permeability are blocked by mepyramine (Schwartz et al., 1986a(Schwartz et al., , 1986b induce breakdown of inositol phospholipids in cerebral cortical slices (Hollingsworth and Daly, 1985), hippocampus (Baudry et al., 1986), A431 human epidermoid carcinoma cells (Hepler et al., 1987). The histamine actions mediated by HP receptors including cardiac dysfunction and gastric acid secretion are blocked by cimetidine which was developed by Black et al. (1972). Stimulation of Hz receptors can induce CAMP accumulation in a variety of tissues including human platelets (Klysner et al., 1980). HS receptors appear to be presynaptic autoreceptors, only recently discovered in brain, which control histamine synthesis and release (Arrang et al., 1985). However, the mechanism of signal transduction via Hz receptors has never been known. Several investigations suggest that histamine modulates the human platelet functions via the histamine receptors (Klysner et al., 1980;Norn et al., 1982). They reported that stimulation of both H1 and HZ receptors reduce the serotonin secretion by thrombin. And Gespach et al. (1986) reported the action of histamine on serotonin uptake by human platelets. They suggest the regulatory role of histamine and the involvement of a new subclass of Hz receptors mediating serotonin uptake.
There is increasing evidence that the breakdown products of inositol phospholipids by phospholipase C, IP$,l and diacylglycerol are important intracellular messengers to play a key role in stimulus-secretion coupling in many tissues including platelets (Berridge, 1984;Nishizuka, 1986). In addition, AA released by phospholipase A2 or its metabolites had functional roles in platelets (Authi et al., 1986;Watson et al., 1986;Kajiyama et al., 1989). In the present study we have investigated the effect of histamine on rabbit platelet functions such as IPs formation, AA release, and serotonin secretion. Here we observed that the addition of histamine stimulated AA release from rabbit (and human) platelets via H1 receptors and enhanced PMA-stimulated serotonin secretion. The activation of phospholipase Az by histamine appears to be mediated by IAP-sensitive G proteins, like other receptor stimulants, thrombin or norepinephrine. However, the mechanism of phospholipase AZ activation by histamine appears to be different from that of thrombin, since the sensitivities to the antiserum against @y subunits of G proteins and to PMA were not equally observed. were from Boehringer-Mannheim. [5,6,8,9,11,12,14, (Murayama and Ui, 1985, 1987bKajiyama et al., 1989;Murayama et al., 1990). Purified G proteins and its 07 subunits were prepared by the methods of Katada et al. (1987). Anti-Gi/G, antiserum and anti-& antiserum were prepared by Dr. Y. Kitamura in this laboratory according to the previous report (Kitamura et al., 1989). Anti-Gi/G, antiserum reacted with a subunits (01~~ and ad1) and p subunits of G proteins. Anti-/37 antiserum reacted with p subunits of G proteins (data not shown, but see Kitamura et al., 1989 (Kajiyama et al., 1989;Murayama et al., 1990). Briefly, platelet-rich plasma was obtained by centrifugation of the blood at 150 X g for 10 min at 20 "C, and platelet-rich suspension was obtained by gel filtration using Sepharose-2B.
Platelets were separated by centrifugation at 1500 x g for 10 min at 20 "C and washed twice with a modified Tyrode Hepes were added to the labeled platelets, and the incubation was continued for 10 min at 37 "C. Thereafter, PMA-treated platelets were precipitated by the centrifugation.
Digitonin, ZAP, and Anti-G Proteins Antiserum Treatment-In several experiments, the labeled platelet suspension was incubated with 10 -15 PM digitonin for 7 min at 37 "C. In Fig. 4, the incubation buffer was further supplemented with 1 rg/ml of the preactivated IAP, 100 pM NAD, 1 mM ATP, 10 mM thymidine, 10 mM dithiothreitol for ADP-ribosylation of membrane proteins. This digitonin treatment was terminated by dilution with 10 ml of the Tyrode buffer, and the permeabilized platelets were sedimented by centrifugation at 1500 x g for 10 min at 10 "C. In Table III, the labeled and permeabilized platelets were incubated for 20 min at 30 "C with 20 bl/ml of anti-Gi/G, antiserum or anti-@y antiserum. fH]AA Release and fH]Serotonin Secretion in Response to Stimuli-The intact or permeabilized platelets were incubated for 7 min to assay for [3H]AA release and for 1 min to assay for [3H]serotonin secretion at 37 "C with 0.1 mM CaCl* and further additions shown in the tables and figures.
In Figs. 2, 3, and 9, these platelets were first incubated with histamine receptor antagonists for 5 min. The reaction was terminated by addition of 1 ml of the ice-cold Tyrode buffer containing 2 mM EGTA and 5 mM EDTA followed by a 2-min centrifugation at 8000 X g at 4 "C. The 3H content of the supernatant was estimated by the liquid scintillation spectrometer. Cyclic AMP Responses of Platelets-The intact platelets were suspended with the Tyrode buffer. Platelet suspensions were incubated for 5 min at 37 "C in the presence of 0.5 mM 3-isobutyl-l-methylxanthine. Further additions are shown in the legend to Table II. Incubation was terminated by acidification with HCl to final 0.1 N and by boiling for 2 min. The cellular CAMP quantitatively transferred to the supernatant was determined by a sensitive radioimmunoassay method (Honma et al., 1977). fHIZP3 Formations of Platelets-Intact platelets were suspended in the Tyrode buffer and incubated for 2 h at 37 "C with [3H]inositol (25 GCi/ml) in the presence of 10 pM indomethacin. The labeled platelets were washed twice with the buffer and incubated for 15 s at 37 "C with additions as shown in Table  I. The incubation was terminated by the addition of 10% trichloroacetic acid, followed by extraction with five washes of 5 volumes of water-saturated diethyl ether. The aqueous phase was applied to Dowex AG l-X8 columns for separation of IP3 as described elsewhere (Berridge, 1984;Kajiyama et al., 1989).

HI Histamine
Receptor-stimulated AA Release- Fig.  1 shows that the radioactivities were increased in the medium when [3H]AA-labeled rabbit platelets were incubated. The rate of release was constant for 10 min in the vehicle-stimulated platelets. The rate of release was enhanced upon the addition of 100 pM histamine. The enhancement was dependent on the concentration of histamine, the half-maximal increase with approximately 2 -5 pM (Fig. 1B). When platelets were preincubated with the indicated concentrations of HI-selective antagonist mepyramine, the stimulatory effect of histamine on [3H]AA release was attenuated in a dose-dependent manner (Fig. 2). No effect was observed by cimetidine, HP-selective antagonist. Table I shows that thrombin provoked rapid formation of [3H]IP3 when added to rabbit platelets within 15 s as previously shown (Kajiyama et al., 1989). Histamine had no effect on IP3 formation.
This finding suggests that histamine-stimulated AA release is mainly due to activation of phospholipase A2 but not to activation of phospholipase C. Klysner et al. (1980) reported that histamine caused an accumulation of CAMP via Hz receptors in human platelets. We examined the effect of histamine on CAMP content (   CAMP accumulation about 1.2-fold. This small effect of histamine was blocked by the addition of 10 pM cimetidine, not mepyramine.
Histamine did not inhibit CAMP accumulation even in the presence of PGE1. The addition of 10 PM PGEl did not stimulate [3H]AA release (data not shown). These data suggest that the observed effect of histamine on AA release is probably mediated by HI subtype of histamine receptors and not by HP subtype and adenylate cyclase systems.
Histamine-stimulated [3H]AA release was also observed in human platelets (Fig. 3). The half-maximal increase was about 0.2 PM, and the effect of histamine was blocked by the addition of 10 FM mepyramine.
These results are in accordance with the previous report suggesting the existence of HI receptors in human platelets (Nom et al., 1982).
G Proteins Interactions with Histamine Receptors and Phos-pholipase AZ--The responses to histamine or other receptor stimulants, such as thrombin and norepinephrine, were examined using permeabilized rabbit platelets (Figs. 4 and 5). Histamine-stimulated [3H]AA release was not observed in the permeabilized platelets, in contrast with intact platelets (Fig.  4C). When the reaction mixture was supplemented with GTP, [3H]AA release was markedly stimulated by histamine. As shown in our previous reports (Kajiyama et al., 1989;Murayama et al., 1990), thrombin-and norepinephrine-stimulated AA release were dependent on the coexistence of GTP (Fig.  4, A and B). Without receptor stimulation, hydrolysis-resistant GTP analog GTP-& evoked the release of [3H]AA in the permeabilized platelets (Fig. 5A). This GTP+-induced AA release was enhanced by histamine. As shown in Fig. 5B, both responses by 10 PM GTP-# in the absence or presence of 100 PM histamine were reduced by the stable GDP analog GDP@. GDP@ inhibited those responses in a dose-dependent manner, and the half-maximal inhibition required about 50 nM, respectively.
Histamine-stimulated AA Release Prevented by ZAP and Anti-G Proteins Antiserum Treatment-Since the above data suggested the involvement of G proteins in AA release by  histamine, the effect of IAP was investigated. IAP, which catalyzes the ADP-ribosylation of certain G proteins such as Gi and G,, is used as a probe to study mechanisms involved in receptor-mediated signal transduction in a variety of cell types (see for review, Ui, 1984;Murayama and Ui, 1985, 1987bNomura et al., 1985). In phospholipase Az assay system using permeabilized platelets, IAP treatment blocked thrombin-, norepinephrine-, and histamine-stimulated [3H]AA releases (Fig. 4). In Table III, the effects of two kinds of anti-G proteins antisera were examined. Pretreatment with anti-Gi/ G, antiserum, which reacted both LY and p subunits of Gi and G,, blocked thrombin-and histamine-stimulated AA release 82 and 92%, respectively. These data suggest that the involvement of IAP-sensitive G proteins in receptors-mediated activation of phospholipase Az including histamine receptors. On the other hand, pretreatment with anti-67 antiserum, which mainly reacted @ subunits of G proteins, blocked the effect of histamine, but not the effect of thrombin. This finding suggests that the participation of G proteins in phospholipase Az activity between thrombin and histamine receptors is not the same manner, in view of the role of P-r subunits, while the exact roles of each subunit (a and P-r) of G proteins is unknown. Enhancement of Histamine-, Not Thrombinand Norepinephrine-, stimulated AA Release by PMA Pretreatment-Phorbol esters such as PMA cause platelet activation at high doses (Rink et al., 1983;Halenda and Rehm, 1987;Saxena et al., 1989). However, several findings indicate that the activation of protein kinase C could induce feedback inhibition of receptor-linked physiological events in platelets (Mobley and Tai, 1985;Zavoico et al., 1985;Watson and Lapetina, 1985;Yoshida and Nachmias, 1987;Murayama et al., 1990). As shown in Fig. 6 and previously reported (Murayama et al., 1990), PMA pretreatment attenuated [3H]AA releases in response to thrombin and to norepinephrine. Histamine-stimulated [3H]AA release, however, was enhanced by PMA pretreatment. When the platelets were simultaneously stimulated by histamine and PMA, the effect of histamine was enhanced about 1.5-fold by PMA addition (data not shown). PMA pretreatment enhanced the maximal response by histamine and increased the affinity for histamine. The EDbO values of histamine were about 10 and 1 pM in control and PMA-treated platelets, respectively. The effect of PMA was dose dependent, an EDSo of 1 nM (Fig. 7), and blocked by the addition of 10 PM 1-(5-isoquinilinylsulfonyl)-e-methylpiperazine (H-7), a protein kinase C inhibitor (data not shown). The stimulatory effect of PMA on histamine-stimulated [3H]AA release was observed when using permeabilized platelets (Fig. 8). The maximal response by histamine plus GTPor GTPyS-stimulated AA release in permeabilized platelets was markedly enhanced, and the affinity for histamine was increased by 10 nM PMA pretreatment. As shown in Fig. 8B, GTPyS-stimulated reaction without receptor stimulants was not reduced in the PMA-treated platelets. The ED5,, values for GTP-S in histamine-stimulated AA release were 1 pM and 25 nM in control and PMA-treated platelets, respectively. These results suggest that the coupling between the histamine receptors and the G proteins leading to AA release, not between the G proteins and phospholipase A*, is positively modified by protein kinase C activation.

PMA-stimulated
Serotonin Secretion Is Enhanced by Histamine Addition-We previously reported that PMA treatment induced both phenomena, stimulation of serotonin secretion at high doses, and diminution of the thrombin-stimulated secretion at relatively low doses (Murayama et al., 1990). Fig. 9 shows that 100 nM PMA pretreatment produced a significant release of the radioactivity from [3H]serotoninloaded platelets in the Tyrode buffer containing 100 pM CaCl*.  The effect of PMA was enhanced by histamine addition in a dose-dependent manner with half-maximal stimulation occurring at approximately 2 wM, while histamine, even if 1 mM used, had no effect on serotonin secretion. The effect of histamine was blocked by 10 pM mepyramine. DISCUSSION

HI Histamine
Receptor, G Proteins, and Phospholipase A2 System in Rabbit Platelets-The results presented herein show, for the first time to our knowledge, that rabbit (and human) platelets have a pathway for H1 histamine receptorsmediated activation of phospholipase AZ and that PMA is a potent stimulator of this histamine-stimulated pathway. There are three types of histamine receptors, the H1 subtype which is coupled to breakdown of inositol phospholipids, the Hz subtype which is coupled to stimulation of adenylate cyclase, and the H3 subtype which is coupled to uptake of histamine in the brain (Schwartz et al., 1986a(Schwartz et al., , 1986b. The following findings suggest that activation of phospholipase AZ is coupled to H, histamine receptors by G proteins in rabbit platelets. 1) Histamine-stimulated [3H]AA or its metabolites release from [3H]AA-labeled rabbit platelets (and human platelets) was inhibited by the addition of H1 antagonist, not by Hz antagonist (Figs. 2 and 3). Histamine did not change the levels of IPB in rabbit platelets (Table I).
3) The histaminesensitive CAMP accumulation, which appears to be uniformly coupled to Hz receptors, was very small (Table II). Under the same experimental conditions, PGE, induced a marked (80fold) increase. Taken together these results show that histamine-stimulated AA release in rabbit platelets is mediated by H, subtype of histamine receptors mainly. A possibility of the involvement of HB subtype receptors is discussed below. 4) In permeabilized platelets, histamine stimulated AA release in the presence of GTP analogs (Figs. 4 and 5). 5) Histaminestimulated release in the presence of GTPyS was antagonized by GDP@ addition (Fig. 5B). 6) Not only thrombin action but also histamine action were reduced by pretreatment with IAP (Fig. 4) and anti-Gi/G, antiserum (Table III). In rabbit platelets, Ca*+ is an important factor to the activation of phospholipase AZ but is not the sole factor to the regulation. G proteins are involved in receptor-mediated activation of phospholipase AZ (Nakashima et al., 1987a(Nakashima et al., , 1987bFuse and Tai, 1987;Kajiyama et al., 1989). These results suggest that IAP-sensitive G proteins are involved in both thrombin receptor-and H1 histamine receptor-mediated activation of phospholipase AZ, Specificity in Signal Transduction of Histamine-The results indicated herein show that the signal transduction pathways activated by histamine that lead to AA release can be distinguished from those utilized by thrombin or norepinephrine. First, the sensitivity for anti-p-y antiserum is different (Table III). Both thrombin-and histamine-simulated AA release were inhibited by IAP treatment and by anti-Gi/G, antiserum treatment. Anti-By antiserum treatment, however, inhibited the histamine action not the thrombin action. The reasons for this difference are not clearly understood. The Pr subunits can modulate the function of several effecters (Neer and Clapham, 1988) including retinal phospholipase AS (Jelsema and Axelrod, 1987). Our results show that the role of subunits of G proteins may be different between thrombin receptors and histamine receptors.
Second, the sensitivity for PMA is different (Figs. 6 and 9). A brief exposure to PMA markedly inhibited thrombin-stimulated AA release (Fig. 6A) and serotonin secretion (data not shown, but see Murayama et al., 1990). The inhibitory effect could be due, at least in part, to uncoupling between receptors and G proteins by activation of protein kinase C (Murayama et al., 1990). In contrast, PMA treatment enhanced histaminestimulated AA release and serotonin secretion. The present findings are somewhat analogous to the observations that PMA inhibits the inositol phosphates production by oil-adrenergic agonists on hepatocyte, but not those by vasopressin and angiotensin II (Cooper et al., 1985;Corvera et al., 1986). Similarly, activation of protein kinase C by PMA results in inhibition of vasopressin-and bombesin-stimulated increases of cytosolic Ca2+ concentration but leaves the platelet-derived growth factor-stimulated response (Lopes-Rivas et al., 1987). The reason for this selective sensitivity for one receptor but not another is not clear. One possible explanation for this effect is a better coupling between histamine receptors and G proteins (Fig. 8). Another possible explanation is the modification of receptor numbers.
PMA is known to modulate several agonists binding to its receptors in many tissues (Motozaki et al., 1986;Bjorge and Kudlow, 1987;Davis and Meisner, 1987). In Swiss 3T3 tibroblasts PMA treatment induced to express more A2 adenosine receptors, not P-adrenergic receptors, enabling binding of the ligand and couple to adenylate cyclase (Murayama et al., 1989). We now measure the radioligand binding for histamine receptors.