MARCO CONCEPTUAL

Furosem ide (700 |iM) reduced anti-IgE stim ulated chloride u p tak e into rat peritoneal m ast cells (Fig. 4.39). MANOVA testing show ed th at this reduction w as statistically significant (Table 4.6). In addition, furosem ide inhibited anti- Ig E -stim u lated h istam in e release by 45.2+4.9 %. W hen the fu ro sem id e concentration w as low ered to 50 pM, the d ru g h ad no effect on anti-IgE- stim ulated chloride uptake (Fig. 4.40, Table 4.6). H ow ever, this concentration inhibited anti-IgE-induced secretion by 16.2±1.7 %.

B u m etan id e h a d no effect on a n ti-Ig E -stim u la ted ch lo rid e u p ta k e at concentrations of 100 pM an d 50 pM, alth o u g h the d ru g p o ten tiated the induced histam ine release by 49.9+17.3 % and 47.8±12.1 %, respectively (Figs 4.41 and 4.42, Table 4.6). Piretanide (100 pM) did not alter anti-IgE-stim ulated chloride uptake and the d ru g had no effect on anti-IgE-stim ulated histam ine release (Fig. 4.43, Table 4.6).

DSCG low ered anti-IgE stim ulated chloride u p tak e at concentrations of 100 pM and 30 pM and inhibited the secretory response by 40.4+4.8 % and 35.3+4.6 %, respectively (Figs. 4.44 and 4.45). MANOVA assessm ent of the attenuated chloride u p tak e u p to 30 m in show ed th a t for b o th co n cen tratio n s the red u ctio n w as not statistically significant (Table 4.6). H ow ever, M ANOVA analysis of the 1 and 10 m in values in d icated th a t the re d u c tio n w as significant for b o th concentrations (100 pM DSCG; F6,i = 6.77, p = 0.041 and 30 pM DSCG; F6,i = 13.85, p = 0.01). This m odification of the data used in the MANOVA assessm ent w as m ade because the m ast cell secretory response is essentially com plete 10 m in after stim ulation.

NPPB (10 pM) significantly reduced anti-IgE-stim ulated chloride u p tak e (Fig. 4.46, Table 4.5) and inhibited anti-IgE-m ediated histam ine release. Fig. 4.47 d e m o n stra te s th a t th e N P PB -m ediated in h ib itio n of an ti-Ig E -stim u lated ch lo rid e u p ta k e an d h istam in e release w ere d o se -d e p e n d e n t o v er the concentration range 5 to 20 pM. Furtherm ore, the a tten u atio n of chloride uptake correlated w ith the m agnitude of the inhibition of histam ine release.

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4.4 DISCUSSION

This stu d y has show n th at furosem ide inhibits anti-IgE-stim ulated histam ine release from ra t p erito n eal m ast cells. The effect is subject to m ark ed tachyphylaxis w h en the cells are p rein cu b ated w ith fu ro sem id e p rio r to stim ulation and in this respect the dru g is similar to DSCG. The tachyphylaxis ex p erim en ts indicate th a t furosem ide and DSCG m ay sh a re a com m on cellu lar target. Both d ru g s h ad som e in h ib ito ry action o n A23187- an d com pound 48/80-stim ulated histam ine release from rat peritoneal m ast cells, alth o u g h these effects w ere only ap p aren t at concentrations of 100 |iM and 1000 pM. Thus, DSCG's and furosem ide's inhibitory action on rat m ast cells appears to be connected to FcgRI receptor activation.

F u ro sem id e in h ib ited concanavalin A -stim u lated h ista m in e release from m ouse peritoneal m ast cells. H ow ever, the IC5 0 value of 300 pM w as m uch

h ig h er th an th a t observed for im m u n o lo g ically -stim u lated ra t p e rito n ea l m ast cells (IC5 0 = 60 pM), suggesting that furosem ide has a less-specific action

on im m unologically-activated m ouse m ast cells com pared to rat p erito n eal m ast cells. In contrast, and consistent w ith previous observations [159], DSCG h ad no effect on concanavalin A -stim ulated histam ine release from m ouse p e r ito n e a l m a s t cells. B oth c o m p o u n d s w e re in a c tiv e a g a in s t im m unologically-stim ulated histam ine release from g u in ea p ig m esenteric m ast cells. In total, this stu d y has dem onstrated th at fu ro sem id e a n d DSCG have a sim ilar inhibitory profile on activated rodent m ast cells.

This stu d y has also clearly dem onstrated that the loop diuretics, furosem ide, b u m etan id e and p iretan id e have distinct m o d u lato ry effects o n activated ro d e n t m ast cells. P iretan id e h ad little effect on an ti-Ig E -stim u la ted rat p e rito n eal m ast cells, w hile b u m etan id e actually p o te n tia te d th e in d u ced h istam in e secretion. It is therefore unlikely th at fu ro se m id e 's in h ib ito ry action on anti-IgE-stim ulated histam ine release is related to th e d ru g 's action on the N a+ /K + /2 C 1 “ cotransport system . B um etanide's m ark e d p o ten tiatio n of the secretory response w as not altered by fu ro sem id e-p retreatm en t of rat peritoneal m ast cells. This im plies th at the tw o loop d iu retics h av e separate cellular targets, w hich have distinct (and opposing) m o d u lato ry effects on rat peritoneal m ast cell secretion.

Furosem ide inhibited anti-IgE-stim ulated chloride uptake into rat peritoneal m ast cells at a h ig h concentration (700 fxM), w hich is co n sisten t w ith a previous study on antigen-induced chloride uptake [483]. H ow ever, w hen the concentration w as reduced to 50 |iM, the d rug had no effect on the stim ulated chloride u p tak e even th o u g h there w as significant in h ib itio n of histam ine release. Typically, furosem ide inhibits the N a + /K + /2 C 1 “ c o tra n sp o rte r at concentrations of ca. 3 |xM [529,543]; therefore the N a+ /K + /2C 1" carrier is probably not involved in anti-IgE-stim ulated chloride u p tak e into m ast cells. The observations th at bum etanide and piretanide h ad no effect on anti-IgE- stim ulated chloride uptake further supports this conclusion.

Previous studies on intact cells have show n that furosem ide inhibits chloride channel activity in the rat lacrymal gland [570] and the frog cornea [571] at the relatively high concentration of 1000 |xM. This m ay therefore account for furosem ide's attenuation of anti-IgE-stim ulated chloride u p tak e at 700 pM. O th er stu d ie s on iso lated ch an n els h av e failed to d e m o n s tra te th a t furosem ide is a chloride channel blocker [543,572] and it has been suggested that the d ru g will reduce chloride conductance in w hole cell preparations as a result of decreasing cytosolic chloride activity [572]. This theory is unlikely to apply to furosem ide's a tten u a tio n of anti-IgE -stim ulated ch lo rid e u p tak e because the reduction reflects a decrease in m em brane p erm eability to the anion, w hich should not be appreciably affected by cytosolic chloride activity.

A n tig en -stim u lated ch lo rid e u p ta k e into rat p e rito n e a l m ast cells w as unaffected by furosem ide (700 |xM) w hen the cells w ere preincubated w ith the d ru g for 5 m in p rior to stim ulation [483]. This observation is interesting as th is stu d y has sh o w n th a t fu ro sem id e's in h ib ito ry actio n on anti-IgE- stim u lated h istam in e release is m ark ed ly red u c ed w h e n th e cells w ere p reincubated w ith the drug. A lthough furosem ide's inhibition of histam ine release does not ap p ear to correlate precisely w ith in h ib itio n of chloride uptake, these findings do show that the two effects m ay be indirectly related.

DSCG (30 and 100 jiM) show ed som e a tten u a tio n of anti-IgE -stim ulated chloride uptake. Inside-out patch clamp studies on RBL-2H3 cells have show n that DSCG is a p o ten t blocker (IC5 0 = 10 p.M) of an tig en -in d u ced chloride

channel activity [482,573]. In these experim ents, DSCG w as applied to the cytosolic side of the m em brane. DSCG is not th o u g h t to cross cell m em branes as it is highly hydrophilic [574] and extracellular application of DSCG has no

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effect on im m unologically-stim ulated m ediator release from RBL-2H3 cells [575]. C onsidering these tw o factors, DSCG chloride channel inhibition m ay therefore be a physiological irrelevance. Certainly, in this study, DSCG h ad a g re a te r effect on a n ti-Ig E -stim u la ted h ista m in e rele ase th a n on th e co rresp o n d in g chloride u p tak e. Thus, the atten u a tio n of ch lo rid e u p tak e could be an indirect effect and a consequence of other actions of the drug.

DSCG's precise inhibitory m echanism of action on rat p erito n eal m ast cells has yet to be determ ined. Early studies on rat peritoneal m ast cells revealed th at DSCG p rev en ted im m unologically-stim ulated ^^calcium u p tak e [576]. The potencies for th e in h ib itio n of histam in e release an d in h ib itio n of 45calcium u p tak e w ere sim ilar. It w as th erefo re p o stu la te d th a t DSCG interacted w ith a calcium channel linked to FcgRI recep to r activation. A recent w hole cell patch clamp study on activated rat peritoneal m ast cells has show n th at DSCG only inhibits non-specific calcium currents an d I^rac high concentrations (200-1000 pM) [577]. This suggests th a t DSCG's m ast cell stabilising effect is not a direct consequence of the d ru g 's action on calcium channels. The same stu d y also show ed that DSCG inhibited cA M P-stim ulated chloride currents at concentrations > 200 |iM. As discussed above, the fact that a relatively hig h co n cen tratio n of DSCG is req u ire d to in h ib it chloride conductance indicates th at the DSCG's potent inhibition of histam ine release from this histam inocyte is a result of other actions of the drug.

A n alternate hypothesis for DSCG's inhibitory action has come from p ro tein phosphorylation studies on rat peritoneal m ast cells [403]. C o m p o u n d 48/80 an d anti-IgE -stim ulation of rat p erito n eal m ast cells resu lts in th e rap id p h o sp h o ry la tio n of th ree p ro te in s w ith in ten s of seco n d s of c ellu lar stim ulation (relative m olecular masses; 42 kDa, 59 kD a an d 6 8 kDa). These

proteins are therefore th o u g h t to be involved in the initiation of secretion. A fourth p ro tein (78 kDa) is phosphorylated 30 to 60 seconds after stim u latio n an d this m acrom olecule m ay be involved in the term in atio n of secretion. DSCG w as found to increase, transiently, the p h o sp h o ry latio n of a 78 kDa protein and dephosphorylation of this protein appeared to parallel th e onset of functional tachyphylaxis in the m ast cell [578]. Therefore, DSCG's action on this p ro tein m ay explain the d ru g 's inhibitory action on rat p erito n eal m ast cells. Recent w ork suggests th at the 78 kDa m acrom olecule is in fact the MARCKS protein, w hich is a specific PKC substrate [442].

Ethacrynic acid h ad a general inhibitory action on secretagogue-induced h ista m in e rele ase fro m ro d e n t m a st cells. P re v io u s stu d ie s h a v e dem onstrated that the d ru g inhibits antigen- and A23187-induced histam ine release from rat peritoneal m ast cells [579,580]. In addition to its action on the N a+ /K + /2C 1“ cotransporter, ethacrynic acid inhibits oxidative and glycolytic ATP pro d u ctio n [449]. M ast cell secretion is associated w ith an increase in cellular A TP-utilisation, therefore ethacrynic acid's inhibitory action alm ost certainly arises from a decrease in ATP production [580]. This w ould account for the d ru g 's non-specific action on secretagogue-induced histam ine release from m ast cells. In addition, any effect of ethacrynic acid on the N a+ /K + /2 C P co tran sp o rt system w o u ld be secondary to the d ru g 's atten u atio n of ATP production.

The chloride channel blocker, NPPB, w as the m ost p o ten t inhibitor of anti- IgE-stim ulated chloride uptake into rat peritoneal m ast cells, suggesting th at ch lo rid e influx occurs th ro u g h ch lo rid e channels. This h y p o th e sis is consistent w ith electrophysiological observations w hich have dem onstrated that NPPB (10 pM) inhibits chloride channel activity follow ing secretagogue stim ulation [481]. M oreover, NPPB's inhibitory action on anti-IgE-stim ulated h istam ine release (IC5 0 = 6 pM) m ay be a consequence of the d ru g 's well

d o cum ented chloride channel blocking action. In su p p o rt of this theory, NPPB-induced attenuation of chloride uptake correlated w ith the m agnitude of inhibition of histam ine release.

NPPB w as fo u n d to in h ib it A23187- an d co m p o u n d 4 8 /8 0 -stim u la te d histam ine release from rat peritoneal m ast cells (IC5 0 values of 8 pM and 9

pM respectively), suggesting th at the d ru g has a rather non-specific m ast cell stabilising action. The results presented in chapter 3 show ed that both A23187- and com pound 4 8 / 80-stim ulation of rat peritoneal m ast cells d id n o t cause significant chloride uptake, although these secretagogues did cause significant histam ine release. C hloride uptake does not ap p ear to be involved in m ast cell h istam in e secretion in d u ced b y n o n-im m unological lig an d s. It w as therefore disappointing th at NPPB-inhibited the response m ediated by these agents. Theoretically, if the dru g 's principal action is on chloride channels, th en m ast cell se cre tag o g u e s th a t do n o t u tilise in cre ased c h lo rid e conductance should be refractory to the effects of NPPB, Electrophysiological stu d ies on rat p erito n eal m ast cells have sh o w n th a t b o th a n tig en and com pound 48/80 induce increased chloride conductances across the m ast cell

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m em brane [405,481]/ so NPPB's attenuation of com pound 4 8 /8 0 -stim u lated h istam ine release is consistent w ith the electrophysiological recordings. It should be noted th at NPPB w as slightly m ore p o ten t at inhibiting anti-IgE- stim ulated histam ine release com pared to the other agents and this m ay, in p art, explain the discrepancies betw een the chloride u p tak e d ata an d the inhibition of histam ine release experim ents.

A stu d y on insulinom a cells found th at NPPB (50 pM) blocked oxidative phosphorylation by acting on m itochondrial anion channels [581]. This effect m ay account for the dru g 's no n specific inhibition of secretagogue-induced histam ine release from rat peritoneal m ast cells. H ow ever, NPPB's inhibitory action was ap p aren t at m uch low er concentrations, and NPPB's inhibition of anti-IgE-stim ulated histam ine release exhibited tachyphylaxis follow ing d ru g p rein cu b atio n , w h ich is n o t co n sisten t w ith the action of an o x id ativ e p h o sp h o ry la tio n u n co u p ler. For exam ple, m ast cell p re in c u b a tio n w ith