An activating effect of bicarbonate on the active transport of Cl- across the rectal wall of Schistocerca gregario has been found, as revealed by an increase of the transmural
potential and short-circuit current. Acetazolamide, an inhibitor of both parameters,
does not show this inhibitory effect, even at much higher concentrations, when
added in the presence of bicarbonate. A Cl- or S O , anion dependent ATPase, different
from the Na+, K+ ATPase, has also been Cound, which can be further stimulated by bicarbonate

Herrera-Mesa, L. (Luis)

Jordana, R. (Rafael)

Lopez-Moratalla, N. (Natalia)

Ponz, F. (Francisco)

Santiago, E. (Esteban)

Universidad de Navarra

Effect of bicarbonate on chloride-dependent transmural potential and ATPase activity in the rectal wall of Schistocerca gregaria

Dadun, University of Navarra

Effect of Bicarbonate on Chloride-Dependent Transmural Potential and ATPase Actlvity in the Rectal Wall of Sehistoeerea gregaria * L. Herrera, N. López-Maratalla, E. Santiago, F. Ponz and R. Jordana Departamento de Zoología, Fisiología y Bioquímica Universidad de Navarra Pamplona (Spain) L. HERRERA, N, LOPEZ-MORATALLA. E. SANTIAGO, F. PON2 and R.  JORDANA. Effect of Bicarbonate on Chloride-Dependent Transmurai Potentiai and ATPase Aaivity in the Rectal Waii of Schistocerca gregaria. Rev. esp. Fisiol., 34, 219-224. 1978. An activating effect of bicarbonate on the active Lranspori of C1- across the rectal wall of Schistocerca Eregarim has heen found, as revealed by an increase of the trans- mural potential and short-circuir current. Acetazolamide, an inhibitor oi both param- eters, does no1 show this inliibirary efiect, even at much higher conceiitrations, when added in the presence of bicarbonatr. A Cl- or S O ,  anion dependent ATPase, dif- ferent from the Na+, K+ ATPase, has alsa been Cound, which can be furthei stimu- lated by bicarbonate. The existence of an active mechanism for transporting C 1  from the lumen to' the haeniolymph across the rectal wall in the desert loilist, Schistocerca gregaria has been already established (2, 3). This transport can be measured by the variations o£ Potential Difference (PD) and sliort-circuit current (Isc) between both compartmeiits: it is also already known how these parameters are affected by metabolic inhibitors as \ve11 as by cer- tain ions. Thus it has been demonstrated * This work was supv~ted by the «Fondo para el Desarrollo de la Investigación Cienti- fica (Presidencia del Gobierna)». that both PD and Isc are directly depen- dent on the active transport of C1- from the luminal side tu the haemolyniph and alsa that the aerobic metabolism is tbe main energy source supporting this active transport. This conclus ion  has heen reached after observing the inhibitioii of PD and Isc in the absence of oxygen, or in the presence of metabolic inhibitors such as KCN or dinitrophenol. Bicarbonate seems tu be implied in this transport mechanism, since a marked in- hibitory effect has been observed on PD and lsc when acetazolamide is present in the medium (3). This effect could be ex- plained in two different ways: 1) That bi- carbonate could be exchanged on the lumi- 220 L. HERRERA, N. I~PEL-MORATALLA, E. S NTIAGO, F. PON2 AND R. JORDANA Table l. Comi,osition of bathing solutions ImMl NaCl 154.8 KCI 12.8 CaCI,GH,O 4.5 MgCI26H2O 4.0 K PO,H, - MgS0,7H,O - Tris-HCI 5.5-5.0 NaHCO, - mina1 side by C1-, and therefore in its ab- sence the entry of C 1  would be hindered, a r  2) Tliat the ti-arisport meclianism of C I  itself were sensitive to bicarbonate. In arder to study which of these two hypotheses could be correct the effect of bicarbonate on PD and Isc has been fol- lowed when this anion was present in the luminal or in the haemocoelic compart- ment os simultaneously in botb compart- ments. The ATPase activity of the tissue was also studied, as well as its relation- ship with the ions participating in this transport. Materials and Methods Adult male and female specimens of the desert locust (Schistocercu greguriu) were used. The way to  obtain the everted rectal preparations and the procedure for ineasur- ing PD and Isc were described in a for- mer paper (2). The solutions have been tliose o l  O'RIORDAN (6) f0r insects and that of KREBS-HLNSELEIT (4) s l igh t ly  modified according to the experiments to be performed (table 1). After a control incubation period of the preparations in thc standard O'Rior- dan medium during approximately 13 min the PD and the Isc were measured. The medium was then rapidly rcplaced in the lumen or in tbe haemocoele or in both compartments b,y the Krebs-Hcnseleit so- Itition (medium with HC0,-) and bub- bling of 95 % O? and 5 % CO, instead of O, with the purpose of maintaining the pN. After 15 minutes of incubation the preparations were then taken back to the initial conditious (O'Riordan medium and 0, bubbling). Acetazolamide when used was always dissolved in Krebs-Hen- seleit medium. The ATPase activity of the tissue has Table II Effect of HCO; and acetazolamide on electric potential differences The change per cent values repiesent the mean of per cenl changes ior each independent ", P -c 0 05 Mean values t S E PD, in mv (Lumen always po5itive) Time íminl  Sfandard Experimental Experimental condi tan Parameter O 13 15 HCO; in Lumen (61 PD 44.2I 4.2 4 2 3 I  3.9 48.2I 3.4 lsO 331.8i46.3 330.9i36.2 383.9k39.6 HC0,- in Lumen and PD 3 1 9 I  4.6 16.4I 2.6 3 1 1 I  1.8 Haemocoele [71 lag 423.0+26.1 218.3i15.1 310.4I 19.1 HC0,- in Haemocoele (71 PD 36.8+ 2.5 26 .0 i  5.4 38.71 5.7 1% 391.7I30.9 299.9t61.7 526.6 126.1 HCO,  and acetazolamide PD 41.3I 3.3 32 .8 I  4.3 40.3I 4.2 1 0 '  M in Lumen (61 Isi 396.3123.8 342.2321 7 458.3 i 36.5 BICARBONATE AND ATPASE IN RECTAL WAl.1 221 been measured according to the following procedure. The rectal walls of three spe- ciinens of Schisrocerca greguria were ho- mogenized in 5 m1 of 0.25 M sucrose, and centrifuged at 14,000 X g for 10 min, and sediments resuspended again in 5 m1 of 0.25 M sucrose; supernatants were dis- carded sincc no aclivity was found in them. ATPase activity was measured de- termining the orthophasphate (1) released from ATP. The incubation medium con- tained in 0.8 ml 5 pmoles of Mg (NO,),. Tris-anions (Cl-, SO,-, HCO,-, NO,-), ph 7.4, at the concentrations indicated in each experiment, an aliquot of the en- zyme suspension (50-100 pg d protein); after an incubation of 5 min at 30" C the enzyinatic reaction was initiated by the addition of 6 pmoles of ATP dissolved in 0.2 m1 of distilled water. After 10 min the reaction was stopped by the addition of 0.1 mi of 50 % trichloroacetic acid. Reagent and enzyme blanks were deter- mined in each experiment. Protein was detennined following the technique of L ~ W R Y  et al. (5 ) .  the addition of bicarbonate in the luini- nal, haeinocoelic compartment or in both compartments, has been studied. After a control incubation period the saline me- diuin of O'Riordan was replaced by that of Krebs-Hanscleit (with bicarbonate), in the luminal or in the haemocoelic side; or in both compartments simultaneously. Aii increase of PD and Isc was observed and their values decreased after returning the preparations to the control medium without bicarbonate (table 11 and fig. 1). The effect of the presence or absence of bicarbonate on PD and Isc was more marked when added in, or when removed from the haemocoelic than from the lum- inal side. This could be explained by a much easier access of the bicarbonate to the cell from the haemocoelic side. Effect of ucetazolamide. It had al- ready been observed in previo'us work (3) that acetazolamide inhibited PD and Isc when present in the luminal side at lo-" and 5 X M concentrations. Siuce acetazolamide is a speci6c inhibitor of carbonic anhydrase iis e f f e c t  was ex- Recults plained as due toa  decrease in intracellular Effect of bicarbonate. The effect of bicarbonate concentration. This seems to and short-circuit currenf strength across the rectal wall of locusf. expe-ent. Student's t test for no independeut values: ', P < 0.001;" P : 0.011 3, P < 0.02; Iaa, in pi cm-'. Number of experiments in parentheses. - and condition condition Standard Change % - Addifion af  Ornission of 30 32 42 HCO. HCO. 222 l.. HBRF33RA, N. L~PEZ-MOFATALLA, E. SANTIh00, F. PONZ AND R. IOBDANA be. confirined with the sesulis here pre- senied which show that at concentrations 10 times higher ¡han those used in pre- 20 vious work, and which caused inhibition Fig. 1 .  Eiectropoieiitial dilference [PD] and shorfkircuit  curreni i isc ]  across the rectal wall of locust. Effect o£ hicarbonate (32.5 mM), or bicarbon- ate and acetazolamide (10 mM) oii lhe lumeii (L), haemocoele (H) or both compartments. PD (e); Isc (O); Control, -; I-IC0,-, Oi HCO; N" L and H a¡ q*--y lo ; \,*-~.~~..L- 4 0 ~  1 lo: measured in ¡he presente of Mg++ added equal to 60 mM the A T P ~ S ~  activiiy did not chaiige. N O ,  anion was found to he much less stimulatory. The ATPase activity found in tlie ho- nlogenates was different from that oF Na+ and K'- ATPase since these cations were 100 of PD and Isc in the absence of bicar- o bonate, no effcct was observed when bi- LO0 carhonate was present iii the medium : ~ O O  either in the lumen or in the haemocoele 2 0 0  (table 11 and fig. 1). - , O 0  30 2" 1 0 ~  0- Table III. Effecf o1 anions on ATPase activity iii homogenates from the rectal wali of Schistocerca gregaria. Number of experiments in parentheses. ATPase activity ' [rnumal ATP hydrolyzedlmg prot x m i n l  O ATPose uctivity. Table 111 shows ¡he effect of anions on ATPase activity of ¡he hanlogenate of the rectum of Schisto- cerca greguriu. The basa1 activity observed was 50 m,u.moles Pi mn-l ~ro te in  min-l as Mg(NO,),. :--a, 1- It may be seen Lhat 60 mM C 1  or 300 60 inM SO,- had a similar effect; their 200 Mgii 15 m M ]  None (15) 5 0 i  4 Cl- 160 mM1 l101 205 i10  SO4= [60 mM1 (10) 200 i10  NO,- 160 mM1 (101 1 0 8 i  5 CI- [30 mM1 + SO4= 130 mM1 (61 204 i11  Cl [30 mM1 + SO,- [10 mM1 (6) 138+ 5 Cl 160 mM1 + N O a  110 mM1 (6) 219 i11  I O eflects were also additive in such a way O 10 20 30 LO that varying the concentraiions of these m," iwo aiiions but maintaining their suin M g l -  [5 mM1 and H C O '  [ ? O  mM1 1 Mean values i S E .  B : C & R B O U r E  AND ATP; O 5 10 ii 23 mM [HCO? 1 Fig. 2. Eiiect of bicarbonate on ATPase acfiv- ify nieasured i!i the presence o i  Ci-. ATPase activity has been expressed as murnoles ATP hydrolyzed min-' . mg-' proteiii. T h i  coii- centralion of CI- was 60 mM. Bars represenl mean values + S.E. Fig. 3 .  Effect of CI- ioii concentrafioi? on ATPase acfivity in fhe presence or absence o i  bicarbonate. ATPase activity has been expressed as mj~rnoles ATP hydralired mir-' - rng-' pratein. Bicar- honiils conciiitialion was always 10 mM (e). In abscissae conceniialioiis «f C l  and NO,- are given in mM. I t  sliould be noticed that the sum 01' ihe concentra(ioiis o [  both ians was kept canstant and equal lo IZO mM. Bars represent mean values k S.E. not uresent in the inedium: however, its activity was sensitive to anions, mainly C 1  dnd SO,- In the ~ccond column of table 111 thc aciivating effect of bicarbonate on ATPase is shown. ATPase activity ineasured in the presence ol either Cl- or SO,- or o£ 7 * \SE I N  RECTAL W~UL 223 botli doubled when 10 niM H C O ,  was added besides those other anions. On the contrary, only a very weak activation took place wiih 10 mM bicarbonate iii the ab- sence of these two latíer anions and re- placed by NO,-. Since the main intraceíiular anion is C1- the effccis of differcní bicarbonate coii- centralions on ASPase activity mcasured in the presence oE 60 mM CI has beeii stndicd, Uiider thesc cond i t i ons  t h e  activity jncreased with iiicreasiiig bicar- bonate coiiceiilrations (O to 20 mM) (fig- ure 2). Figure 3 shows the cifect of clifferent concentrations of C1-, maintaining un- chaiiged the ionic strength o£ the inedium ivith NO,-. T i  may be seen that a linear relationship was found belween ATPase activity and C I  concentraíion within the range of concentrations studied. If 10 mM H C O ,  \vas also preseiit a new enhaiice- mcnt of ATPase activity took place. Role of HC0,- on PD and Isc. It had already been poiníed out in a previous repoi-t (3) that HCO,- could play an im- portalit rolc in the flux of ions from the luinen to the haeinocoele in the rectal wall of Sch. ~reguriu, after thc obscrvation that aceíazolamide produces an inhibitioii of PD and Isc when present i i ~  tbe incuba- tion medium. The dala here presented give furthcr support lo tlie vicw that bi- carbonate may play a key role in the transport of CI-. The presence of bicar- bonate in thc mediuin bathing lhe luminal sidc enhanced the PD and the Isc: it might be lhought íhat this cffect could be due to a flux of HCO,- from the lumen to Ihe hacmocoele and causing therefore an increase of PD and Isc. Howcver, lhis explanation is not valid since the same effect was observed when the bicarbonate was simultaneously preseiit in the lnminal and jn the haemocoelic side, or only in the haeinocoele. It might also be thought 224 L HERRERA, N LOPEZ MORATALLA, E SANTIAGO, F PONZ AND R JORDANA that the bicarbonate effect could be ex- plained by an exchange of H C O ,  by CI- in the luminal side, taking place in this way the entry of Cl- into the cell. If this werc the case the effect due to bicar- bonate should be much lower whenl b'i- carbonate were present both in the lumen and in the Iiaemocoele. Since the effect was thc same regardless the compartinent where the bicarbonate was added, and even when added io both compartments, this hypothesis can also be discarded. Be- sides, when acetazolamide, which inhibit- ed PD and Isc in the absence of bicar- bonate, is added in the medium at con- cenlrations 10 times higher than those necessary to causc inhibition, no inhibi- lory effect was observed if bicarbonatc was present in the bathing solutions. Thus, it secms reasonablc to think that the observed effect of bicarbonate on the PD and Isc, and consequently on the ac- tive transport of C1-, is a direcl effecl on the mechanism of this transport. Tt is pos- sible to think that some potential changes may have arisen from junctions poten- tials, because the solutions which are used in both sides (lumen and haemocoele) are different b'ut we have carried out exper- iments which prove that using identical solutions on E'oth sides (O'Riordan solu- tion) the addition of bicarbonate in the presence of acetazolamide in the haemo- coele, in the lumen o'r in both sides, al- ways increased the transmural potential, and in that case there was no possibility of the existence of junction potentials. ATPase activify. She existcnce of an ATPase in the tissue of the rectal wall dependent on the presence of CI-, and activated by bicarbonate, could very well be related t o  the transport mechanism of C1- ions into thc cells, since, as discussed above, C I  and HC0,- affect the PD and Isc in the rectal sac of Schistocerca gre- guria. Resumen El ion bicarbonato produce un incremento del potencial transmural y de la corriente de curtocirciiita cn la pared rectal de Schistocerc<i gregnrin. La acelazolamida que inhibe ambos parámetros n o  produce efecto alguno -inclusa a alias ~ o n c e n t r a c i o n e s ,  en presencia de b i ~  carbonato. Se delemina una ATPaia depen- diente de aniones ( C l  o SO,,-) diferente de la ATPasa Naf, K+. Esta ATPasa dependiente de aiiiones se estimula por e l  bicarbonato. l .  FISKE, C. II. and S~BBAROW, Y.: l. Bioi. Clzem., 66, 375-400, 1925. 2. HERRTRA, L., JOKDANA, R. and PONZ, F.: J .  Z17.secI Phyrii~l., 22, 291-297, 1976. 3. HERREM. L., JORDANA, R. and PONZ, F.: J.  Znscct Physiol., 23, 677-682, 1977. 4. KREBS, H. A. and HENSELEIT, K.: Z. P h y ~  siol. Chem., 210, 33~66, 1932. 5. LOWRY, O. H., ROSEBRO~GH, N. J., FARR, A. L. and R ~ A L L ,  R. T. :  J. Riol. Chem., 193, 266-275, 1951. 6. O'RIORDAN, A. M.: l. Exp .  Biol., 51, 699- 714, 1969. 

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Effect of bicarbonate on chloride-dependent transmural potential and ATPase activity in the rectal wall of Schistocerca gregaria

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