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Sodium transport across the locust rectum Black, Kenneth Thomas 1983

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SODIUM TRANSPORT ACROSS THE LOCUST RECTUM by KENNETH THOMAS BLACK B.Sc. UNIVERSITY OF BRITISH COLUMBIA, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1983 © KEN BLACK 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Z O O L C ^ V T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e DF-fi ( 2 / 7 9 } - ii -A B S T R A C T It was the purpose of this study to further elucidate the movements of N a + 22 across the rectal epithelia of locusts, Schistocerca gregaria. The kinetics of Na absorption are investigated yielding a K m of 17.2 mM and a V m a x of -2 -1 1.54 u Eq cm h . The effects of a stimulant (cAMP) and inhibitors (Amiloride and 22 Ouabain) are also investigated. cAMP (1 mM) has no effect on Na flux but does + + 2 1 cause an increase in short-circuit current (I ) (1.28 - 0.12 to 4.80 - 0.28 uEq cm" h" ) sc n and potential difference (P.D.). (4.4 - 0.3 to 13.2 "tj.8 mV) at a N a + concentration of 22 55 mM. Amiloride (1 mM) causes a 3 3 % reduction of Na influx and a 7 5 % reduction 22 of net Na absorption by the recta. The inhibition caused by amiloride is reversible. 99 n Ouabain has no effect on Na flux at room temperature (21 C) but causes a 3 7 % 22 reduction of Na influx (lumen to hemocoel) at 29 C. Ouabain and amiloride have no effect on I or P.D. Extracts from several neuroendocrine tissues had no conclusive sc 22 effect on Na fluxes as tested. - iii -TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS ii TABLE OF FIGURES iii INTRODUCTION 1 METHODS 7 RESULTS 13 Viability of Unstimulated Preparations 13 and Time to Steady-State Flux Across Stimulated Recta 20 Kinetics of Transepithelial Na Transport 25 Across Short-circuited Recta 22 Effect of Inhibitors on Na Fluxes 33 Across Short-circuited Locust Recta Effects of Extracts from Neuroendocrine Tissues 40 • on N a + Absorption by Locust Recta DISCUSSION 44 BIBLIOGRAPHY 51 - iv -TABLE OF FIGURES PAGE TABLE 1 COMPOSITION OF SALINE 11 FIG. 1 DIAGRAM OF USSING CHAMBERS 12 FIG. 2 I AND UNIDIRECTIONAL 3 6C1" FLUXES 14 sc FIG. 3 UNIDIRECTIONAL AND NET 2 2 N a + FLUXES 16 FIG. 4 2 2Na FLUXES OF INDIVIDUAL PREPARATIONS 18 FIG. 5 AVERAGE I WITH TIME ACROSS UNSTIMULATED RECTA 21 sc FIG. 6 UNIDIRECTIONAL FLUXES AND I IN RESPONSE TO cAMP 23 SC TABLE 2 2 2Na FLUXES, I AND P.D. AT VARIOUS ' sc EXTERNAL Na + CONCENTRATIONS 28 FIG. 7 UNIDIRECTIONAL 2 2 N a + FLUXES AS A FUNCTION OF EXTERNAL Na + CONCENTRATION AND KINETICS OF NET Na + FLUX 29 FIG. 8 HANES-WOOLF PLOT OF Na + TRANSPORT KINETICS 31 FIG. 9 WOOLF-AUGUSTINSSON-HOFSTEE PLOT OF Na + TRANSPORT KINETICS 34 FIG. 10 EFFECT OF OUABAIN ON FORWARD 2 2 N a + FLUX ACROSS INDIVIDUAL PREPARATIONS 37 TABLE 3 EFFECT OF NEUROENDOCRINE TISSUES ON 2 2 N a + INFLUX 42 - V -ACKNOWLEDGEMENTS I would especially like to thank Dr. J.E. Phillips for his support, assistance and patience. I would also like to thank Joan Martin for her assistance and fellow students John Hanrahan, Mary Chamberlin and Kevin Strange for their support. I also thank Dr. V. Palaty for his guidance through the past several years. -1 -I N T R O D U C T I O N Much work has been done to characterize ion transport across the locust rectum. Ion transport in this organ is important for maintaining and regulating hemolymph ion levels. This is of particular importance for a small terrestrial insect like the locust because of its large surface-to-volume ratio and because the arid environment in which the locust lives imposes a severe stress on the insect's osmoregulatory abilities. A major regulatory problem is the conservation of body water. Water is reabsorbed from the rectal lumen against large (i.e. 1 osmolar) osmotic differences (reviewed by Maddrell 1977, Phillips 1977 a, b). At same time, hemolymph ion levels must be regulated during this water absorption and also during ingestion and elimination of excess water when insects feed. Phillips (1964) showed that, in Schistocerca fed hypertonic salt solutions or fresh water, the hemolymph ion concentrations remain relatively constant. This is also the case when hemolymph volume is reduced by more than 50% as a result of dehydration (Hanrahan, 1978; Chamberlin and Phillips, 1979). The role of the excretory system in maintaining hemolymph composition in insects has been reviewed frequently in recent years (Wall and Oschman, 1975; Phillips, 1977, 1980, 1981; Maddrell, 1980). Initially an isosmotic urine is secreted by the Malpighian tubules. This then flows into the rectum, via the hindgut, where a selective reabsorption of ions, organic metabolites and water occurs. The major ions recycled through the excretory system are K + , C l " and to a lesser extent N a + . The concentrations of these ions in primary urine entering the rectal lumen are 99 to 139mM K + , 20 to 46mM N a + and 93 to llOmM C l " , whereas the hemolymph concentrations are 11, 108 and 115mM respectively (Phillips, 1964). Hanrahan (1982), - 2 -Spring (1980) and Williams et al (1978) have characterized C l " and K + transport in the recta of locusts in some detail using short-circuited preparations and flux measure-ments. Hanrahan (1982) utilized a saline based on the measured ionic composition of hemolymph from S. gregaria. He also compensated for the series resistance of the saline for the first time during short-circuit studies of this epithelia. Williams et al (1978) and Spring (1979) made measurements of N a + , K + and C l " transport rates at one external concentration but they used a less effective saline and they did not compensate for saline resistance so that significant errors were possible. They also made their measurements during the first 4 h after excising the recta so that some residual hormonal stimulation remained. Hanrahan's studies were all conducted during 4 to 8 h after excision of this tissue. Hanrahan (1982) determined that C l " absorption from the rectal lumen was the major ion transport process after cAMP stimulation. This anion transport is also stimulated by luminal K + but is independent of external N a + . Na coupled co-transport of C l " , which has been demonstrated in many vertebrate tissues (reviewed by Frizzell et al , 1979), is apparently not the mechanism of C l " transport in this tissue (Hanrahan and Phillips, 1983a). In support of this view, removal of N a + from the bathing solution had no effect on C l " transport. The inhibitor of Na-K-ATPase, ouabain, which should dissipate the apical N a + electrochemical gradient, and furosemide, which inhibits the coupled Na-Cl co-entry step, did not reduce C l " transport even at ImM concentrations at 22°C. Hanrahan studied the kinetics (K and V ) of K + and C l " transport across m max r the recta but not of N a + . Spring (1979) and later Hanrahan (1982) demonstrated that - 3 -cAMP or corpora cardiaca (CC) extracts cause larqe (3 to lOx) increases in I and sc ' ^ C l net flux across locust recta. Spring showed that this treatment had no effect on 22 + net Na transport at an external Na concentration of 80mM. This again suggests N a + absorption is not coupled to movements of KC1 in this tissue. Indeed, Phillips has pointed out there may only be enough N a + entering the locust rectum to drive reabsorption of amino acids, phosphate and acetate by co-transport mechanisms (Pillips, 1982). N a + movement across most absorptive epithelia is a two step process (Leaf, 1965). Entry of N a + into the cell across the mucosal boundary is usually passive. In vertebrates it is thought that N a + often enters through a Na- selective site which is sensitive to amiloride, or by exchange for H + or NH^. It is not clear if the same mechanism occurs in insect epithelia. This is followed by the active extrusion of N a + at the serosal membrane by a Na-K -ATPase , i.e. the "Sodium Pump". Komnick and Achenback (1979) and Ernst et al (1980) have shown, by utilizing 3(H)-ouabain binding, that the pump is located on basolateral surfaces of epithelial cells in dragonfly recta and frog skin respectfullly. This primary N a + pump at the serosal border maintains the internal cellular sodium concentration at very low levels, thereby ensuring a strong inward electrochemical gradient for passive entry of N a + across the mucosal membrane. For example, in Periplanta recta, the local sodium concentration in the lumen is 36mM, in the cytoplasm is 28mM and in the hemocoel is 145mM (Wall and Oschman, 1975). So sodium readily enters the epithelium from the lumen passively. A negative intracellular potential (not measured in this species) would further enhance -4 -the entry greatly. Using ion-sensitive electrodes, Hanrahan (1982) found that in Schistocerca the net electrochemical potential difference across the mucosal mem-brance of recta was 127mV, with PD of 64mV, (hemocoel negative) thereby providing a large driving force for Na + entry. Some of the passive Na + entry is coupled to the entry of neutral amino acids in vertebrates (reviewed by Schultz, 1977) and also in locusts (reviewed by Phillips, 1980). Balshin (1973) estimated that up to 20% of Na + absorption in locust rectum might be accounted for by coupled amino acid entry. However, since this estimate proline absorption has been found to be much greater than he anticipated. Indeed proline and Na + levels in fluid entering locust recta in situ are similar (i.e. 30-40 mM; Chamberlin, 1981). A coupled Na- glycine entry mechanism has been clearly shown for this tissue (Balshin, 1973). "^C-Glycine influx obeys Michaelis-Menten kinetics (K = 22mM at a Na + concentration of 174mM). If all the Na + is replaced with choline, the influx is drastically reduced (reviewed by Phillips, 1980). Phosphate and acetate are actively absorbed in the locust rectum (Andrusiak, 1974; Baumeister et al 1981) and these anion transport processes may be coupled to Na + absorption in a similar manner to amino acids. Some Na + absorption may occur in exchange for cellular H + or NH^ since there is evidence that both these cations are secreted into the locust rectal lumen (Hanrahan, 1982; Speight, 1967). Thus Na + absorption might be implicated in pH regulation in terrestrial insects such as the locust. - 5 -In this study I investigate the kinetics of transepithelial sodium transport in locust recta and the effect of cAMP, which is known to stimulate I . I also report on the effects of amiloride and ouabain on sodium fluxes and I . Benos et al (1979) state sc that amiloride does not act on "leaky" epithelia. As revealed by electrical studies (Hanrahan and Phillips, 1983a) locust recta exhibit properties that would classify it as 2 a "tight" epithelia with low transcellular resistance (i.e. 100-200 XI cm ) as compared to frog skin, on which most work with amiloride has been done, which is a "tight" epithelium of high resistance ( 10,000 Si cm ). Amiloride inhibits the passive sodium entry step (Bentley, 1968). Liedtke and Hopfer (1977) indicated that H +/Na + exchange can be inhibited in many tissues with amiloride. So the effects of amiloride on ion transport across locust recta is of interest. Hanrahan (1982) found that ImM ouabain had no effect on I across cAMP-stimulated recta at room temperature. This sc r was not surprising because I is due to Na-independent C l " transport. However, Peacock (1981) showed that a ouabain-sensitive Na +-K +-ATPase is present in locust rectal tissue (Kj = 10~^M). However, it was possible that during experiments by Hanrahan (1982) ouabain did not penetrate through the outer tissue layers to an ATPase on basolateral membranes or that the low experimental temperature reduced ouabain inhibition of such an ATPase. The effect of ouabain on the preparations at higher temperatures and for longer exposure times in this study re-investigates previous reports that ouabain has no effect on I and net Cl flux across locust recta. This is important because it is one line of evidence that Cl" entry is not coupled to Na + entry (Hanrahan, 1982). - 6 -Since hormonal control of Na reabsorption in insect excretory systems has not been studied, I investigated the effects of extracts from locust neurosecretory tissues on rectal Na + flux. Tolman and Steele (1980 a, b), in studies on Periplanta recta, suggest that there are CC factors which may act on monovalent cation transport although they did not measure fluxes. A heat-sensitive antidiuretic factor stimulates oxygen consumption and short-term fluid reabsorption from an isosmotic sugar solution bathing these recta, but only if Na + is present on the hemocoel. These processes are also ouabain-sensitive. They suggest that a putative natriferic hormone from the retrocerebral complex increases Na + movement from the hemocoel to Na + transport sites on the lateral membranes. This results in increased of fluid absorption supported by Na + transport. A systematic study of all the major neurosecretory tissues should show if such a natriferic factor is present in locusts. - 7 -METHODS Adult female Schistocerca gregaria that were 14-30 days past their final moult were used in all experiments. Females were used because of their larger size. These animals were raised at 28°C and 60% relative humidity on a 12:12 h light: dark cycle and fed fresh lettuce daily. They were also fed a dry mixture of alfalfa, bran and powdered milk. Recta were excised from the locusts and mounted as flat sheets between two modified Ussing chambers (Williams et al 1978, Hanrahan, 1982). The recta were stretched over eight tungsten pins on a raised collar and secured with a rubber O-ring (see Fig. 1). There is negligible edge-damage using this method (Hanrahan, 1982). Both sides of the tissue were bathed with identical salines which were bubbled vigorously with 95% 0^ and 5% CC>2' This ensured rapid and complete mixing of the saline. The tissue was short-circuited with compensation for series resistance of the saline (circuitry modified from Rothe et al 1969, by Hanrahan, 1982). Short-circuit current (I ) was monitored on a Soltec 220 recorder. Open circuit potential SC difference (P.D.) was recorded periodically and before every flux sample by briefly stopping I for 10 sec. This brief period of open circuit had no visible effect on I sc sc during fluxes, since the I was identical before and after the open circuit, and was too 22 + short to significantly alter Na flux measurements. Various salines used to bathe the recta are shown in Table 1. Control saline was based on an analysis of locust hemolymph by Hanrahan (1982) and Chamberlin (1981). - 8 -When reducing Na + concentration (by removing NaCl), appropriate amounts of choline chloride were added to maintain C l " concentration at 105 mM. Sodium methyl sulphate was used to increase the Na + concentration beyond 110 mM. When required, the tissue was stimulated by adding 50 ul of a 101 mM stock solution of cyclic adenosine monophosphate (cAMP) to both sides of the chamber bringing the final concentration of cAMP to 1 mM. 22 + When using ouabain, Na fluxes were measured before and after addition of ouabain to the saline. A 50 ul aliquot of ouabain from a stock solution was added to each side of the chamber to bring the final concentration of the inhibitor to 1 mM. Normal saline was used during these investigations. Amiloride presented special problems because of its low solubility. Amiloride (1 mM) was dissolved in control saline that lacked any sulphates (Amiloride saline Table 1). After flux measurements were made in the presence of normal saline, this saline was replaced completely with a similar saline containing the 1 mM amiloride. To check if the effects of amiloride were reversible, the preparations were initially bathed in the saline containing 1 mM 22 + amiloride. After the Na fluxes were measured, the saline was replaced twice completely with normal saline and fluxes remeasured in the absence of this inhibitor. Amiloride was a gift from W.D. Dorian, Merck Frosst Laboratories. Ouabain, cAMP and all amino acids were obtained from Sigma. All salts were reagent grade. 22 + To investigate effects of neurosecretory tissues on Na absorption, extracts of brain, pars intercerebellis, suboesophageal ganglia, thoracic ganglia 1 to 3 and - 9 -abdominal ganglia 4 to 8 were added to the mucosal side of short-circuited recta. Two flux measurements were taken before and three after addition of each extract. The extracts of locust neuroendocrine tissues were prepared in our lab by J. Proux (Personal Communication). Whole glands or ganglia were excised from female locusts and were then sonicated in normal saline for 10 min. The resulting mixture was centrifuged (10,000 g for 5 min) and the supernatant containing water-soluble neuropeptides were tested as described above at dosages of 0.1 to 3.2 glands in the 5 ml of saline bathing the serosal side of recta in Ussing chambers. 22 To measure transepithelial Na fluxes, Na (New England Nuclear, carrier free) 22 was added as NaCl in aliquots of 20 ul from stock isotope solution to one chamber. This side will be referred to as the "hot side". After a short period of mixing, duplicate 20 ul samples were taken from the "hot side" and placed in vials containing 1 ml of "cold" saline. These samples were used to determine the radioactivity of the "hot side". To determine the increase in radioactivity of the "cold side", 1 ml samples were taken at intervals of 15 or 30 minutes and were replaced in the chamber with equivalent volumes of "cold" saline. All samples were counted on a gamma counter (Model 1058 Nuclear Chicago). Unidirectional flux was calculated using the following formula (Williams et al 1978): a 2 V C Jl-2 = a ^ T A -2 -1 where: ^ *s the unidirectional flux (uEq cm h" ) a l is the radioactivity of the "hot side" (cpm ml"^) a -1 2 is the increase in radioactivity of the "cold side" (cpm ml ) C is the concentration of the unlabelled ion in solution (mM) V is the volume of the solution in the chambers (5 ml) A is the tissue surface area (0.196 cm ) T is the time interval between samples (h) - 10 -Fluxes, simultaneous I , and periodic P.D. measurements were a l l made 2 to k h after removing recta from locusts and under conditions similar to those used by Hanrahan (1982), unless otherwise indicated. Unless otherwise mentioned, the temperature was 21°C. When temperature was increased in later experiments, this was achieved using a variable heater, with thermostat. The experimental protocol and the ranges of I and P.D. considered acceptable for in vitro rectal preparations were similar to those of Hanrahan (1982) to permit comparison of results. The lower acceptable limits for I and P.D. were SC -2 -1 1.0 uEq cm h and 3.0 mV respectively (preparations unstimulated). Preparations that f e l l below these values were discarded. A l l errors are expressed as plus or minus the standard error of the mean. 11 Table 1(a) Salt Composition of Salines (mM) Used to Bathe Recta NaCl K2SO^ KC1 MgS0 4 7H zO Mg(N0 3) 2 6H 20 NaHCO, CaCl 2 2H 20 Na- Methyl SO^ Choline Chloride Amiloride Glucose Sucrose Normal Saline 100 5 10 10 5 10 100 Low Na Saline Varied 5 10 10 5 Varied 10 100 High Na + Saline 100 5 10 10 5 Varied SO. Free Saline (for Amiloride study) 100 10 10 5 10 100 1 10 100 Alanine Asparagine Argenine Glutamine Glycine Histidine Lysine Proline Serine Tyrosine Valine Table Kb) Amino Acids (mM) in all Salines of (a) 2.91 1.31 1.00 5.00 11.37 1.41 1.44 13.14 6.50 1.87 1.78 - 12 -FIGURE 1 USSING CHAMBERS a. calomel electrodes (C) connected to 1.5m KC1 agar bridges (B). Saline was circulated and oxygenated via gas lift pumps (G). - 13 -RESULTS Viability of Unstimulated Preparations and Time to Steady-State Initial measurements of ^ C l " fluxes were carried out across short-circuited recta in normal saline during the 2nd to 4th h after dissections to determine if the procedures yielded results similar to those previously obtained by Hanrahan (1982) who used identical methods. For unstimulated preparations, the forward " ^ C l " flux, lumen (L) to hemocoel (H), and back-flux (H to L) were 3.11 - 1.21 and 2.53 - 0.13 uEq cm'^h"^ respectively, yielding an average net flux of -2 -1 0.58 uEq cm h (n=7). The average I and P.D. during these flux measurements *f" 2 1 + were 1.41 - 0.29 uEq cm" h~ and 7.1 -0.6 mV (H-side negative) and values for L to H and H to L fluxes were not significantly different. A l l these values agree closely with previously published values for unstimulated recta under identical conditions (Hanrahan 1982). Since I and P.D. conformed closely to previous data, these two transport parameters were subsequently used to assess the viability of the rectal preparations, because forward and back fluxes of necessity were measured on different preparations. The time course of typical I and " ^ C l " fluxes appears in Fig. 2. Steady state values were approximated after the first hour. 2 2 Transepithelial Na fluxes were measured in a normal saline (110 mM Na ) across the short-circuited, unstimulated preparations to determine the period required 22 + for Na to equilibrate with the tissue pool of Na . After 30 minutes flux values were constant (Fig. 3). Therefore, fluxes were subsequently measured between one half and two hours after the addition of 2 2 N a , and two to three hours after dissection. Fig. 4 - 14 -Fig. 2 -2 -1 The change in I (uEq cm h ) with time after dissection for typical rectal preparations while monitoring ' ^ C l ~ fluxes. The " ^ C l " fluxes across some individual unstimulated rectal prepara-tions. ^ C l was added to one side of the chambers 1 h after dissection. L to H #,0,A,A ; H to L • ,• V unidirectional fluxes. - 15 -Fl G.2 8 b T 1 - i 1 r - 16 -22 The average Na fluxes L to H • • , H to L • o , and net flux with time, to determine the period required to reach steady-state. Normal saline contained 110 mM Na +. Na was added to the saline 4 hs after dissection in absence of stimulants. - 18 -F J g ^ 22 Na fluxes for some individual unstimulated preparations to show variability of flux with time in any one preparation. L to H A,•,0,V5 H to L •, •, •, v 1 conditions as in Fig. 3. - 19 -- 20 -shows the fluxes with time for several individual unstimulated preparations, indicating that there was not a great deal of variability with time after the f i r s t flux period for any one preparation, compared to that between preparations. Steady-state forward flux (L to H) was 4.68 - 0.65 uEq cm'V 1 and the back-flux (H to L) was 2.43 - 0.19 uEq cm~ 2h - 1. Net flux was 2.25 uEq c m " 2 h - 1 from L to H (n=8). The I ^ sc and P.D. during measurements of Na from L to H were 1.58 - 0.24 uEq cm'^h"^ and •f* 22 4.9- 1.3 mV. Where Na flux was measured H to L, the I was ' sc 1.49 - 0.14 uEq cm'V^ and P.D. was 5.1 - 0.6 mV. The similarity between these values indicate that preparations used to determine forward flux behaved similarly to those used to determine back-flux. These values agreed closely with those reported -2 -1 by Hanrahan (1982; 1.47 uEq cm" h" , indicating that anions were moving to the hemocoel side, and 5.04 mV, H-side negative.) A time course of I and P.D. for a l l SO preparations is shown in Fig. 5. Flux Across Stimulated Recta 22 The effect of cAMP on Na flux was investigated because this causes several fold increases in I g c , P.D., C l " flux and K + flux. These experiments were done in conjunction with experiments on the kinetics of N a + movement discussed below. With external N a + concentration at 55 mM, the steady-state forward flux (L to H) was 1.54 - 0.10 uEq cm~ 2h~^ before stimulation with c AMP and 1.55 - 0.25 uEq cm~2h~^" after addition (n=8). Likewise, back flux (H to L) did not change significantly, being 1.31 - 0.11 uEq c m " 2 h _ 1 before and 1.35 - 0.09 uEq c m ~ 2 h _ 1 after addition of cAMP. 22 The effect of cAMP on unidirectional Na fluxes across some individual preparations 22 is shown in Fig. 6. These results indicate that c AMP has no effect on Na fluxes as was previously reported by Spring et al (1978) using somewhat different - 21 -Fig. 5 Shows the I with time after dissection across unstimulated recta in sc + 22 normal saline containing 110 mM Na and during Na flux studies. i s c • — m , P . D . o — o - 23 -Fig. 6 22 Unidirectional Na fluxes before and after the bilateral addition of 1 mM cAMP to some individual preparations to show the variability with time and between individual preparations. cAMP was added at 22 + the arrow, 45 minutes after the addition of Na : L to H V J T J O , * ; H t o L fluxes. The response of I of some individual preparations showing the effect of cAMP during experiments in a). 1 mM cAMP was added at the arrow, 5 h after dissection. - 24 -F I G . 6 0 - 25 -conditions, i.e. his saline contained 55 mM N a + and lacked most amino acids, and he made no correction for series resistance of the saline. I and P.D. showed the sc expected increases after the addition of cAMP, as reported by Spring et al (1978) and Hanrahan (1982). I and P.D. increased from 1.28 - 0.12 uEq c m " 2 h - 1 and sc ^ 4.4 - 0.3 mV to 4.80 - 0.28 uEq cm'V 1 and 13.2 - 0.8 mV respectively (n=8). Similar increases in I and P.D. were observed at other external N a + levels and fluxes (Table sc 2). Fig. 6b shows the time course of I in some individual preparations during this flux studies. Kinetics of Transepithelial Na Transport Across Short-circuited Recta Some i n i t i a l attempts to investigate N a + transport kinetics were done on unstimulated preparations. The N a + concentration was increased by adding sodium methylsulphate bilaterally from a concentrated stock solution. This method caused a drop of up to 2 5 % in I and P.D. as N a + concentration was increased by 30 mM. Preparations run at a single N a + concentration showed higher I and P.D. than when the same SC concentration of N a + was achieved by stepwise addition of sodium methylsulphate. 2 2 4* 2 1 At 55 mM the forward Na flux (L to H) was 1.54 uEq cm" h" and the back -2 -1 flux (H to L) was 1.31 uEq cm h . The I and the P.D. were 1.28 - 0.12 uEq cm~ 2h~^ and 4.4 - 0.31 mV respectively. However, when this N a + concentration was attained by adding suitable amounts of Na methyl SO^ to an + 2 1 initially N a + free saline, the I s c and P.D. dropped from 1.31 t 0.20 uEq cm" h" and - 26 -3.7 - 0.6 mV to an I of 0.91 - 0.15 uEq cm'V 1 and 2.1- 0.6 mV respectively after the N a + concentration was raised. This drop in I and P.D. was below the acceptable 22 + limits outlined in the methods. Since c AMP has no effects on Na fluxes at 55 and 110 mM Na +, the kinetics of Na transport were subsequently determined only for cAMP stimulated preparations. The N a + concentration was raised two or three times for each preparation by adding Na methyl-SO^ bilaterally and the experimental conditions were overlapped to check variability between runs; e.g. one group of preparations were run at sequentially increasing N a + concentrations of 30, 85, and 140 mM and another group at concentrations of 20, 55, and 120 mM. 2 2 + + The Na fluxes at various Na concentrations appears in Table 2a. The back-flux was a linear function of external N a + concentration. The curve for H to L flux was fitte d by linear regression analysis with a resulting correlation coefficient (r) or 0.996. Both the forward and net fluxes showed a non-linear increase (fig. 7) consistent with Michaelis-Menten kinetics. These values of net N a + flux were confirmed using variations of the Michaelis-Menten equation: V S _ max V ~ S + K m where: V = J ^ 3 max max = Na concentration max ,Na K = Na concentration at k maximum J m max - 27 -Table 2(a) 22 Na fluxes at various external Na concentrations under short-circuited conditions (x - SE, n = 6-8) 2 2 N a + Fluxes Na Concentration (uEq.cm" h"l) (mM) (L to H) (H to L) Net 10 0.80 ± 0.18 0.35 ±0.10 0.55 20 1.26 - 0.25 0.43 ±0.10 0.83 30 1.72 ±0.26 0.67 ±0.15 1.05 50 2.27 ±0.23 1.20 ± 0.09 1.07 65 3.12 ±0.66 1.87 ±0.26 1.25 85 3.28 ±0.20 1.58 ± 0.36 1.69 110 3.95 ± 0.50 2.78 ±0.38 1.17 120 4.06 ± 0.56 2.09 ±0.39 0.97 140 4.22 ±0.56 3.78 ± 0.33 0.44 Table 2(b) I and P.D. during flux measurements in a sc a Na + Concentration (mM) -2 -1 I (uEq.cm h~ ) sc P.D. (mV) 10 13.02 t 1.92 26.6 ± 4.6 20 11.94 ±0.78 25.0 ±2.9 30 14.09 ± 1.41 32.6 ±2.4 50 13.31 ± 2.23 27.4 ±4.1 65 11.67 ±0.88 24.1 ± 3.1 85 14.72 ± 1.49 32.8 ±2.1 110 10.25 ±0.87 18.4 ±1.8 120 10.87 ±1.22 22.6 ±3.3 140 13.73 ±1.31 31.3 ±2.4 - 28 -F i g , ? 2 2 Unidirectional Na fluxes as a function of external Na concentration under short-circuit conditions at 20° C. Forward flux L to H Back flux H to L . Line for H to L flux was fitted by linear regression analysis with a corelation coefficient of 0.996. The curve showing kinetics of net N a + flux with changing N a + concentration, is the difference between mean unidirectional fluxes in a). - 2 9 F I G . 7 1 1 1 i 1 1 1— 20 40 CO 80 100 120 140 EXTERNAL N a + CONCENTRATION (mM) - 30 -Two plots were used to determine the K . A Hanes-Woolf plot (Dixon and Webb, m r 1958) which reduces errors since it does not concentrate values of 1/S at one end of -2 -1 the line. With this plot, the K was 17.14 mM with a V of 1.54 uEq cm h . The m max line was fitted to the data by linear regression analysis with a correlation coefficient (r) of 0.991 (Fig. 8) The second plot was a Woolf-Augustinsson-Hofstee plot (Dixon and Webb, 1958). This plot reduces errors due to 1/V. Again the line was fitted to the data by linear regression analysis with r = 0.951. The K was 17.2 mM and the V was 3 m max -2 -1 1.54 uEq cm h (Fig. 9) in good agreement with value from the Hanes-Woolf plot. 22 Effect of Inhibitors on Na Fluxes Across Short-Circuited Locust Recta I studied the effects of two well known inhibitors of Na absorption in 22 vertebrates, amiloride and ouabain, on Na fluxes across locust rectum. Amiloride acts by blocking the Na entry step (either Na channel or Na+/H+ exchange) at the apical membrane, whereas ouabain acts by inhibiting the Na-K ATPase located on the basolateral borders of the cells and therefore the active exit step for Na+. 22 Steady-state Na fluxes were measured before and 1 h after the addition of 1 mM amiloride while the preparations were stimulated with cAMP. The Na+ 22 concentration of the saline was 110 mM. The forward Na flux decreased from 4 42 + 0 33 to 2 95 - 0.51 uEq cm'^ h""'' after adding this inhibitor (n=8). The back flux + 2 X did not change noticeably; 2.42 - 0.18 before and 2.46 - 0.35 uEq cm" h" after adding 22 amiloride. The decrease in Na flux from L to H was 33% and significant at «* =0.05. 22 -2 -1 More noticeable was the drop in net Na flux from 2.00 to 0.49 uEq cm h . This drop in active net Na absorption represents a 75% inhibition caused by amiloride. - 31 -Fig^ B Shows a Hanes-Woolf plot which plots the external N a + concentration 2 2 divided by the net Na flux against the N a + concentration. The line was fitte d using linear regression analysis giving the correlation coefficient of 2 1 0.991. The Vmax is equal to the slope of the line, 1.54 uEq cm h , and the K m equals the ^ m a x multiplied by the y-intercept, i.e. 17.14 mM. - 32 -F I G . 8 100 80-1 10 ?0 30 40 50 60 70 80 9*0 Na* CONCENTRATION (mM) - 33 -22 + A Woolf-Augustinsson-Hofstee plot of the net Na flux against the net 2 2 + + Na flux divided by the external Na concentration. The line was fit t e d by linear regression analysis with a resulting correlation coefficient of -0.951. The K is equal to the negative of the slope, 17.2 mM and the -2 -1 V " m a x is equal to the y-intercept, 1.54 uEq cm h . - 35 -In a separate series of experiements the inhibition caused by amiloride was found 22 + 2 1 to be reversible. The forward Na flux increased from 3.35- 0.55 uEq cm h in the presence of amiloride to 4.70 - 0.83 uEq crn^h"''' when the saline was replaced with one lacking amiloride (n=8). This was a 2 9 % increase in influx and again is significant at the 5% confidence level. Amiloride had no effect on I or P.D. Before addition of amiloride to cAMP sc ry "1 stimulated recta, I was 10.25 - 0.87 uEq cm h and the P.D. was 18.4 - 1.8 mV. ' sc ^ One hour after the addition of amiloride, the I and P.D. were respectively 10.61 ± 0.87 uEq cm" h" and 18.9 - 1.9 mV. In the second set of experiments when the preparations were initi a l l y bathed in amiloride, the I was 2 1 7.96 - 0.82 uEq cm h and the P.D. was 18.7 - 1.4 mV. After the amiloride was •4* 2 1 removed, the I was 8.11 - 0.77 uEq cm" h" and the P.D. was 18.5 - 1.1 mV for the sc ^ same preparations. These differences were not significant. These results substantiate earlier reports that electrogenic C l " transport in locust rectum, which is responsible for virtually a l l I after stimulation, is not coupled to Na transport. Amiloride does appear to inhibit transepithelial Na movement, but the mechanism of inhibition is unclear. 22 + The effects of ouabain on Na flux were not so conclusive. The preparations were not stimulated with cAMP and were bathed with a saline containing 110 mM Na +. 22 2 1 The Na fluxes before the addition of 1 mM ouabain were 3.85 - 9.75 uEq cm" h~ (L to H) and 2.39 - 0.18 uEq cm" 2h" 1 (H to L) (n=8). Sixty minutes after the addition + 2 1 of ouabain the forward flux had dropped somewhat to 3.53 0.68 uEq cm" h" , whereas 2 1 the backflux (H to L) stayed the same at 2.36 - 0.21 uEq cm" h" . However, this - 36 -average drop in the influx (L to H) is not significant. In all but one run, the N a + forward influx was slightly lower after the addition of ouabain (Fig. 10) but this drop is s t i l l insignificant when analysed with a paired t test. The I and P.D. did not change after the addition of ouabain in agreement with earlier observations by Hanrahan (1982). Before the addition of ImM ouabain to the + 2 X unstimulated preparations, the I was 1.77 - 0.19 uEq cm" h" and the P.D. was + * + 2 1 5.2 - 0.4 mV, and after addition of this inhibitor the I was 1.79 - 0.13 uEq cm" h~ ' sc ^ and the P.D. was 5.1 - 0.5 mV. A recent review by Anstee and Bowler (1979) indicates that Na transport in most insect epithelia is unusally insensitive to ouabain. These workers have shown that ouabain inhibition is very temperature-dependent. Temperatures well above room temperatures are often necessary to observe an effect with ouabain. Indeed ouabain inhibition of Na-K-ATPase isolated from locust rectum is neglible at room tempera-ture and only 5 0 % at 32° (Donkin and Anstee 1980). Moreover, the cuticle and basal tissue layers on the mucosal and serosal sides respectively of locust rectal epithelium may act as diffusion barriers, thereby slowing the access of ouabain to the basolateral c e l l border, where Na-K-ATPase has been located in recta of dragonfly larvae (Komnick et al 1979). To overcome this diffusion problem, preparations were stimulated with cAMP and exposed to 1 mM ouabain for longer periods (3 h), with the anticipation that this should give ouabain ample time to enter the tissue. During these experiments, the temperature was initially elevated to 35° C to enhance ouabain - 37 -Fig. 10 22 + The forward Na fluxes (L to H) across some individual short-circuited rectal preparations, before and after the bilateral addition of 1 mM Ouabain. 1 mM ouabain in normal saline lacking cAMP was added to the preparations at the arrow. The saline contained 110 mM N a + and the temperature was 21° C . - 38 -- 39 -inhibition of Na-K-ATPase. However, under this condition the preparations did not remain viable for more than three hours as judged by a steady decline in I and P.D. As a compromise, the temperature was reduced to 29-30° C. At this temperature the preparations appeared viable for at least six to seven hours as judged by stable I and P.D. Donkin and Anstee (1980) report 4 5 % inhibition of isolated locust rectal 22 Na-K-ATPase at this temperature. I found the forward Na flux f e l l in 60 minutes 2 1 from 3.32- 0.50 uEq cm" h~ before the addition of ouabain to 2.11 - 0.36 uEq cm~ 2h"^ after its addition (n=6). This was a 36.5% decrease in active 22 Na influx (L to H) and is statistically significant by a standard t test (©<=0.05). Again I and P.D. did not change significantly after the addition of ouabain. The I g c 2 1 and P.D. were 11.96- 0.38 uEq cm" h" and 17.5 - 0.5 mV before adding ouabain and 2 1 11.423 0.95 uEq cm" hi and 18.5 - 1.5 mV at one hour after adding this inhibitor. After a 3 h exposure to 1 mM ouabain at 30° C. the I was s t i l l 10.85 - 1.52 uEq cm'V 1 and the P.D. was 17.5- 2.5 mV. This slight decline in I and sc P.D. is similar to the normal decay of I and P.D. with time in the absence of ' sc ouabain. In conclusion, at, 30° C ouabain causes partial inhibition of Na transport approaching the reduction predicted from studies with the isolated Na-K-ATPase from this tissue. Again lack of effect of ouabain on I and PD confirm the conclusion by Hanrahan (1982) that C l " transport (i.e. I ) is independent of Na transport. - 40 -Effects of Extracts from Neuroendocrine Tissues on N a + Absorption by Locust Recta Since insect hormones which influence body N a + levels and epithelial transport have not been reported, except for some preliminary and indirect evidence by Steele et al (1980), the effects of extracts from the major locust neurosecretory tissues on 22 + 22 + rectal Na flux was also investigated. Na influx was selected because it includes both active and passive components so that a change in either could be detected, and then the specific component identified later if necessary. An in i t i a l test was run using extracts from the brain, the sub-esophageal ganglion, pars intercerebralis,the thoracic and abdominal ganglia. The results appear in Table 3. Short-circuited rectal preparations were bathed in normal saline containing 110 mM Na at 2 1 u C. The 2 2 Na influxes were measured both before and after measuring dose response curves of I to the extract (by J. Proux unpublished observation), so that maximum amounts 22 of gland extracts were present during the second Na flux measurement. -41 -Table 3(a) The Effect of Neuroendocrine Tissues on ZTsla + Influx Across Short-circuited Locust Recta Gland Accummulated Dose Flux (L to H)(uEq.cm~ h~ ) (8.2 gland-equivalents/5ml) Before After Net Change Brain (n=4) 2.16 ± 0.43 2.27 ±0.40 +5% Sub-esophageal(n=3) 3.58 ± 0.62 3.36 ± 0.45 -6% Pars Intercerebrallis (n-3) 2.84 ±0.39 2.62 ± 0.60 -8% Thoracic Ganglia 1-3 (n=3) 3.11 ±0.74 5.45 ±0.23 +75%* Abdominal Ganglia 4-7 (n=3) 4.10 ±1.03 3.70 ± 0.90 -10% Abdominal Ganglia 8 (n=3) 3.56 ±0.18 2.60 ±0.10 -27% Thoracic Ganglia 1 (n=3) 2.23 ±0.52 2.64 ±0.30 +19% Thoracic Ganglia 2 (n=3) 2.27 ±0.21 2.94 ± 0.40 +30%* Thoracic Ganglia 3 (n=3) 2.67 ±0.15 2.96 ±0.29 +11% Thoracic Ganglia 1-3 (n=3) 110 mM N a + 2.49 ±0.23 2.38 ±0.24 -4% * significant atc<*0.05 Table 3(b) Before After I sc PD 'sc PD Brain 6.0 + 0.6 7.2 ± 1.3 22.0 ± 5.5 15.7 ± 2.5 Sub-Esophageal 9.1 + 0.5 9.8 ± 1.5 14.5 ± 1.5 14.0 ± 0.7 Pars Intercerebrallis 6.0 + 0.9 9.0 ± 0.9 13.0 ± 2.1 15.3 ± 1.7 Thoracic Ganglia 1-3 6.4 + 0.4 3.6 ± 0.3 23.5 ± 1.6 14.5 ± 1.5 Abdominal Ganglia 4-7 3.5 + 0.5 6.0 ± 1.0 5.0 ± 0.9 8.0 ± 0.8 Abdominal Ganglia 8 4.0 + 0.6 4.1 ± 0.6 8.0 ± 1.2 8.4 ± 1.1 Thoracic Ganglia 1 6.3 + 0.7 6.2 ± 1.1 9.5 ± 2.1 7.7 ± 1.7 Thoracic Ganglia 2 7.3 + 0.8 7.0 ± 0.6 10.3 ± 1.2 8.0 ± 1.5 Thoracic Ganglia 3 8.3 + 0.9 10.3 ± 1.8 15.7 ± 1.8 19.7 ± 1.8 TG 1-3, 3 Glands 110mMNa + 6.7 + 0.4 3.8 ± 0.4 13.2 ± 1.1 7.2 ± 0.9 TG 1-3, 3 Glands 20mMNa + 8.5 + 1.5 6.5 ± 1.4 18.3 ± 1.9 12.3 ± 1.3 -42 -22 + were present during the second Na flux measurement. Only the thoracic ganglia extracts showed a possible effect i n i t i a l l y , that of increasing in N a + influx by 75%. Therefore, the effect of this neuroendocrine tissue was studied in more depth. These further tests were done with a pooled extract of thoracic ganglia 1, 2, and 3. The equivalent of three ganglia were added to the hemocoel side of the recta bathed with 5 ml of saline containing 20 mM Na +. A lower Na concentration was used in case a natriferic factor might change K rather than V of active Na transport. 3 m max r The forward Na fluxes before and after the addition of extract were 0.87 - 0.12 and + 2 1 + 0.85 - 0.13 uEq cm" h respectively (n=8). This result shows that at a Na concen-tration near the K for N a + net flux, which is also a typical N a + value observed in the m 22 + rectal lumen in situ, extracts of thoractic ganglia had no effect on the Na influx. The same experiment was repeated with a saline containing 110 mM Na +. Again 22 + there was no significant change in the forward Na flux. Before the addition of thoracic gland extract the 2 2 N a + flux (L to H) was 2.49 - 0.23 uEq c m " 2 h _ 1 and after + 2 1 it was 2.38 - 0.24 uEq cm" h~ (n=6). These more extensive results suggest that 22 + extracts of thoractic ganglia have no effect on Na influx, in contradiction to results in a few preliminary experiments. The I and P.D. values during experiments with extracts from neuroendocrine tissues appear in Table 4b. - 43 -DISCUSSION The purpose of this study was to characterize sodium absorption under appro-priate in vitro conditions, similar to those employed by Hanrahan (1982) in his study of KC1 absorption across locust recta. He concluded that electrogenic C l " transport was the main active process in this tissue after stimulation with CTSH (Chloride Transport Stimulating Hormone) or cAMP, which is the second messenger of CTSH. This enhanced anion absorption is coupled to passive absorption of K +. He provided extensive evidence that C l " transport was not coupled to N a + absorption or HCO^ secretion (Hanrahan and Phillips, 1983a) as i t is in vertebrate epithelia ( F r i z z e l l et al 1979). In this study additional evidence for the lack of Na-coupled C l " transport in locust recta was provided. There was considerable evidence for active N a + transport by locust rectum prior to this study but the kinetics and magnitude of this process were unknown. During salt deprivation, N a + is absorbed from the lumen of ligated recta in situ against larger concentration differences (i.e. tenfold) than can be explained by the transepithelial potential (T.E.P.) of about 20mV (lumen positive; Phillips, 1964). In the absence of external K + and C l " , active N a + absorption, even from dilute solutions, sustained fluid transport across everted rectal sacs at low rates, against N a + concentration differen-ces and in the absence of a sizeable TEP (Phillips et al 1982). These results suggested a low K t for N a + transport. Moreover, ImM ouabain inhibits fluid transport by 5 0 % across everted rectal sacs (Irvine and Phillips, 1970) although incubation -44 -conditions were clearly sub-optimal in these early experiments (see Phillips 1982). Both Williams et al (1978) and Spring and Phillips (1980b) measured a net flux of 2 2 N a + -1 -2 from lumen to hemocoel across short-circuited locust recta (4.4 and 2 u Eq h" cm" and with flux ratios of 4:1 and 2:1 respectfully). However, these authors did not correct for the resistance of the saline bathing the recta and this saline was physiologically less satisfactory than that used by Hanrahan (1982). There measurements were also made at a single high external N a + concentration. In this present study, correcting for saline resistance and using Hanrahan's saline, I obtained a 22 + somewhat lower J for net Na flux under short-circuited conditions (about 1.5 max - 1 - 2 -uEq h cm over the 2nd to 4th h). This is about 1 0 % of J for active C l ^ max absorption under the same conditions (lOmM external K +; Hanrahan and Phillips, 1983a). Results in this study confirm a report by Spring and Phillips (1980b) that 22 + cAMP and CTSH did not affect Na fluxes under 'near' short-circuited conditions. It is now well established for most absorptive epithelia, especially in vertebrates, that sodium enters passively down a favourable electrochemical gradient due to a low intracellular level of N a + and a favourable P.D. across the mucosal membrane. This situation is maintained by extrusion of N a + from the c e l l across the serosal membrane against high N a + concentration and P.D. due to Na +-K-ATPase. There is indirect evidence for such a mechanism in locust rectum. As in many other epithelia, the rectal c e l l interior of locusts and other insects is negative to the outside (Phillips, 1964; Vietinghoff et al 1969; Spring et al 1978; Hanrahan and Phillips, 1983a, b). The intracellular concentration of N a + is lower than that of external fluids bathing - 45 -locust recta (Phillips, 1964, 1980) and this has also been confirmed by microprobe analysis for Calliphora rectal pads (Gupta et al 1980). Hanrahan et al (1982) used double-barrelled ion-sensitive microelectrodes to measure intracellular N a + activities and e l e c t r i c a l potential. He was thus able to calculate electrochemical differences across apical and basolateral c e l l borders of locust recta exposed to varying luminal N a + activities and with transepithelial P.D. clamped at OmV. Intracellular N a + levels remained between 3 and 15mM under a l l conditions and there was a favourable electrochemical gradient for passive N a + entry when luminal N a + levels exceeded ImM. In Hanrahan's saline (i.e. HOmM Na +) the net electrochemical difference favouring passive entry (mucosal) and active exit (serosal) was very large (127mV). Hanrahan noticed no effect of ImM ouabain on these gradients at room temperature unpublished research). (This apparent inconsistency can now be explained by observations made in this study.) Finally, Hanrahan (1982) demonstrated active accumulation of K + inside rectal epithelial cells to a control level of 70mM when K-depleted recta were re-exposed to lOmM K + on the hemocoel side. This is again consistent with a N a + - K + exchange pump on the serosal membrane. A ouabain sensitive (kC 10~^M) Na-K-ATPase with biochemical properties similar to those observed in other tissues has been found in recta of locusts and other insects (Peacock, 1977, 1981; Komnick and Achenbach, 1979). In the case of the ATPase from locust rectum, lOOmM N a + and 20mM K + are needed for maximal activity. Komnick and Achenbach (1979) used ^(H)-ouabain and autoradiography to localize the Na-K-ATPase at the basolateral c e l l border of rectal epithelial cells in -46 -dragonfly larvae, but this location has not been re-investigated for terrestrial insects. Donkin and Anstee (1980) argue that the failure to demonstrate ouabain inhibition of Na +-dependent transport processes in many insect epithelia was because experiments were conducted at room temperature. They showed that ouabain inhibition of Na-K-ATPase from locust is neglible at room temperature, is only 4 5 % at 30°C, and is similar to that of vertebrate Na-K-ATPase at 37°C. In the present study, I found that 22 + ouabain only inhibited active influx of Na if the room temperature were raised to 29°C, at which point the inhibition of N a + flux was similar to that of Na-K-ATPase 22 + reported by Donkin and Anstee (1980) (i.e. 36%). When Na fluxes were inhibited by ouabain there was no effect on Cl~-dependent I which reconfirms Hanrahan's r sc conclusion that electrogenic C l " transport in locusts is not coupled to N a + transport. This idea is further supported by the experiments with amiloride. This agent inhibits 22 + active net Na flux without affecting Cl"-dependent I . Finally, varying luminal SC Na levels in the present study did not change Cl-dependent I across stimulated recta during kinetic studies. 22 + Demonstrations of partial inhibition of net Na flux across locust rectum by ouabain and amiloride provide additional evidence for a mechanism of N a + transport similar to that found in vertebrates. NaCl co-transport generally occurs in "leaky" vertebrate epithelia where the transepithelial P.D. is too small to drive C l absorption by electrical coupling, which occurs in tight epithelia ( F r i z z e l l et al 1979). Since locust rectum is effectively tight (Hanrahan, 1982) it is not surprising that anion and cation absorption are not directly coupled by a co-transport process (Hanrahan and -47 -Phillips, 1983a, b). What is unusual in this tissue is that an electrogenic C l " transport is the predominant transport process and that K + is the major counter ion absorbed after stimulation. Amiloride inhibits N a + entry into other epithelia via selective channels (e.g. frog skin, Benos et al 1968; rabbit colon, F r i z z e l l and Turnheim, 1978) or by facilitated exchange for H + (Liedtke and Hopfer, 1977). The dose-response relationship for amiloride inhibition differs by two-to-three orders of magnitude for these two entry processes (Warnock, et al 1982). Only a high dosage of amiloride was used in the present study, so it is not possible to distinguish between the two types of entry processes which may be inhibited in this tissue. However, other observations have some bearing on this matter. Hanrahan (1982) found that lowering luminal N a + levels from 110 to less than ImM had no significant effect on the mucosal P.D., indicating a very low conductive permeability of this membrane to N a + (P^a-1-)' The mucosal P.D. was largley a K + diffusion potential (55mV change per decade change in luminal K +) with a smaller electrogenic component associated with the apical C l " pumps (about lOmV). In support of low P ^ g of the apical membrance and high P^, Goh (reported in Phillips, 1980) found that tissue N a + was lost largely to the serosal side, whereas tissue K + was lost to the lumen side when locust rectal sacs were bathed bilaterally in an isosmotic sucrose solutions. Thus N a + entry into this tissue from the lumen probably occurs largely by coupled mechanisms. Active absorption of neutral amino acids (Balshin, 1973, Balshin and Phillips, 1971), acetate (Baumeister et al 1981) and phosphate (Andrusiak, 1974) have been shown in locust rectum. In the case of glycine - 48 -the transport process is clearly coupled to N a + (Balshin and Phillips, 1971; Balshin, 1973). The lumen side is acidified by an active mechanism in situ (Speight, 1967, Phillips, 1964) and across unstimulated recta in vitro (Thompson, Personal Communication). The magnitude of this H + secretion is 0.4 to 1.4 uEq fT^cm" 2. If this occurs by N a + - H + exchange at the apical border, i t would account for a large portion of the net N a + entry into rectal epithelial cells of the locust. Future work might be to investigate the H + secretion and the effects of amiloride on H + movement to discover i f it is comparable to N a + inhibition observed as a result of this drug. It might also prove interesting to discover if amiloride has any effects on amino acid movement thereby confirming the coupling of N a + transport to amino acid transport. The low « t for net Na +-absorption in the locust rectum (17mM Fig. 8 and 9) is similar to values for other vertebrate epithelia. The K^ . for Na +-absorption is 22mM in toad urinary bladder (Ussing et al 1974), lOmM in frog skin, Rana pipiens, (Cereijido et al 1964), and 44mM in rabbit urinary bladder (Lewis and Diamond, 1976). A l l of these values were determined while the epithelia were short-circuited. These tissues are all "tight" epithelia; i.e. only a small percentage of ion flow occurs via a paracellular pathway. These "tight" vertebrate epithelia all exhibit transepithelial resistances 2 greater than 10,000 A cm (Lewis and Diamond, 1976). When stimulated with 2 cAMP, locust recta have lower trancellular resistances (50-150 It cm ) but only 5 % of current flow is by the paracellular route (Hanrahan, 1982). Another difference between locust rectal epithelia and vertebrate epithelia is that Na +-substitution has a dramatic effect on I across these vertebrate epithelia (Lewis and Diamond, 1976) but sc not locust rectum. -49 -It has been shown that c A M P and CTSH did not affect N a + fluxes even though I 3 sc and ^ C l ~ fluxes increased (Spring and Phillips, 1980b). These authors only tested the actions of other neuroendocrine tissues from locusts on I and not on N a + fluxes. If sc these endocrine extracts acted on a N a + absorption process which is electroneutral, their effect would not have been detected. Tolman and Steele (1980a, b) have provided some indirect evidence for a natriferic factor in C C . of cockroaches, which enhances fluid reabsorption by rectal sacs. Prior to this study there were no systematic surveys of insect endocrine organs for factors directly influencing N a + reabsorption across insect recta or indeed any aspect of N a + regulation in insects. Using procedures similar to those for extraction of several water-soluble insect neuropeptide hormones, I was unable to detect any factor influencing rectal N a + absorption, although thoracic ganglia gave mixed results which perhaps warrant further study (Table 2). It was in the thoracic ganglia that Proux (1981) isolated a vasopressin-like hormone. It is s t i l l possible that a locust natriferic factor exists which is either very unstable or is not extracted by the procedures used for hydrophilic polypeptide hormones. This possibility should be investigated. SUMMARY 1. The kinetics for Na flux across the unstimulated locust rectum are established -2 -1 with the Km being about 17.2 mM and V of 1.54 uEq cm h . 3 m a y ^ 2. It is verified that c A M P has no effect on the N a + flux across recta. - 50 -Amiloride is found to inhibit Na flux from lumen to hemocoel by 3 3 % and net Na absorption by 75%. The effect of amiloride is reversible. 2 2 Ouabain (1 mM) has no effect on Na flux at room temperature (21°C) but at higher temperatures (29°C) there is a 3 7 % inhibition of Na influx from lumen to hemocoel. Extracts from several neuroendocrine tissues had no conclusive effects on the fluxes of N a + across the locust rectum. - 51 -BIBLIOGRAPHY Andrusiak, E.W. Resorption of Phosphate, Calcium, and Magnesium in the in Vitro Locust Rectum. M.Sc. Thesis, University of British Columbia, Vancouver, Canada, 1974. Anstee, J.H. and K. Bowler. Ouabain- sensitivity of insect epithelial tissues. Comp. Biochem. Physiol. A 62: 763-769, 1979. Balshin, M. Absorption of Amino Acids in Vitro by the Rectum of the Desert Locust Schistocerca Gregaria. Ph.D. Thesis, University of British Columbia, Vancouver, Canada, 1973. Balshin, M. and J.E. Phillips. Active Absorption of amino acids in the rectum of the desert locust (Schistocerca Gregaria). Nature London 233: 53-55, 1971. Baumeister, T., J. Meredith, W. Julien, and J. Phillips. Acetate transport by locust rectum in Vitro. J. Insect Physiol. 27: 195-201, 1981. Benos, D.J., L.J. Mandel and R.S. Balaban. Mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia. J. Gen. Physiol. 73: 307-315, 1979. Bentley, P.J. Amilordie: A potent inhibitor of sodium transport across toad bladder. J. Physiol. 195: 317-330, 1968. Cereijido, M., F.C. Herrera, W. Flanigan and P.F. Curran. The influence of Na concentration on Na transport across frog skin. J. Gen. Physiol. 47: 879, 1964. Chamberlin, M. Metabolic Studies in Locust Rectum. Ph.D. Thesis University of British Columbia, Vancouver, Canada, 1981. - 52 -Chamberlin, M.E. and J.E. Phillips. Regulation of Hemolymph free amino acids in the desert locust. Fed. Proc. 38: 970, 1979. Donkin, J.E. and J.H. Anstee. The effect of temperature on the ouabain-sensitivity of N a + - K + - a c t i v a t e d ATPase and fluid secretion by the Malpighin tubules of Locusta. Experimentia 36: 986-7, 1980. Dixon, M. and E.C. Webb. The Michaelis Constant, in Enzymes, Longmans, Green and Co., London pp. 19-21, 1958. Ernst, S.A., C V . Riddle and K.J. Karnaky Jr. Relationship between localization of N a + - K + ATPase, cellular fine structure, and reabsorptive and secretory electrolyte transport in Current Topics in Membrance and Transport (F. Browner, A. Kleinzeller and E.L. Boulpaep, eds.) Academic Press, London. Vol. 13 pp. 355-385, 1980. F r i z z e l l , R.A., M. Field and S.G. Schultz. Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol. 236(1): F1-F8, 1979. F r i z z e l l , R.A. and K. Turnheim. Ion transport by rabbit colon: II. Unidirectional sodium influx and the effects of Amphotericin B and Amiloride. J. Mem. Biol. 40: 193-211, 1978. Gupta, B.L., B.J. Wall, J.L. Oschman and T.A. Hall. Direct microprobe evidence of local concentration gradients and recycling of electrolytes during fluid reabsorption in the rectal papillae of Calliphora. J. exp. Biol. 88: 21-47, 1980. Hanrahan, J.W. Hormonal regulation of chloride in locusts. The Physiologist 21: 50, 1978. Hanrahan, J.W. Cellular mechanisms and regulation of KC1 transport across an insect epithelium. Ph.D. Thesis, University of British Columbia, Vancouver, Canada, 1982. - 53 -Hanrahan, John and J.E. Phillips. Mechanisms and control of salt absorption in locust rectum. Am. J. Physiol. 244: R131-142, 1983 Hanrahan, J.W. and J.E. Phillips. Cellular mechanism of KC1 absorption in insect hindgut. J. Exp. Biol. In Press. 1983b. Irvine, H.B. and J.E. Phillips. Effects of respiratory inhibitors and ouabain on water transport by isolated locust rectum J. Insect Physiol. 17: 381-393, 1970. Komnick, H. and U. Achenbach. Comparitive biochemical histochemical and autoradiographic studies of Na +/K +-ATPase in the rectum of dragonfly larvae (odonata, aeshnidae). Eur. J. C e l l Biol. 20: 92-100, 1979. Leaf, A. Transepithelial Transport and its hormonal control in toad bladder. Ergebn.  Physiol. 56: 216-263, 1965. Lewis, S.A. and J.M. Diamond. N a + transport by rabbit urinary bladder, a tight epithelium. J. Membrane Biol. 28: 1-40, 1976. Liedtke, C M . and U. Hopf er. Anion transport in brush border membranes isolated from rat small intestine. Biochem. Biophys. Res. Commun. 76: 579-585, 1977. Maddrell, S.H.P. Insect Malpighian tubules. In Transport of Ions and Water in Animals (B.L. Gupta, R.B. Moreton, J.L. Oschman, B.J. Wall, eds.), pp 541-570 Academic Press, London, 1977. Maddrell, S.H.P. Characteristics of epithelial transport in insect Malpighian tubules. In Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.) Academic Press, London, Vol. 14 pp 427-463, 1980. Peacock, A.J. Distribution of N a + - K + activated ATPase in the hindgut of two insects, Schistocerca and Blaberus. Insect Biol. 7: 393-395, 1977. - 54 -Peacock, A.J. Further studies of the properties of locust rectum N a + - K + ATPase with particular reference to the ouabain sensitivity of the enzyme. Comp.  Bioc. Physiol. 68C: 29-34, 1981. Phillips, J.E. Rectal absorption in the desert locust, Schistocerca Greqaria Forskal II. Sodium, potassium and chloride. J. Exp. Biol. 41: 39-67, 1964. Phillips, J.E. Excretion in insects: function of gut and rectum in concentrating and diluting the urine. Fed. Proc. 32: 2480-2486, 1977a. Phillips, J.E. Problems of water transport in insects. In Water Relations in Membrane  Transport in Plants and Animals. (A.M. Jungreis, T. Modges, A.M. Kleinzeller, S.G. Schultz, eds.) pp 333-353 Academic Press, New York. 1977b. Phillips, J.E. Epithelial transport and control in recta of terrestrial insects. In Insect  Biology in the Future (M.L. Locke and D.S. Smith, eds. pp 145-177, New York: Academic, 1980. Phillips, J. Comparitive physiology of insect renal function. Am. J. Physiol. 241: R 241-257, 1981. Phillips, J.E. Endocrine control of salt and water balance: Excretion. In Endocrinology  of Insects (H. Lauder and R. Downer eds.) Alan R. Liss, New York. 1983. Proux, J. Regulation neuroendocrine de la diurese chez le criquet migrateur. Doctoral thesis, L'Universite de Bordeaux, France. 1981. Rothe, C.F., J.F. Quay and W.M. Armstrong. Measurement of epithelial e l e c t r i c a l characteristics with an automatic voltage clamp device with compensation for solution resistance. I.E.E.E. Trans. Bio-Med. Engin. BME-16(2): 160-169, 1969. Schultz, S.G. Sodium-coupled solute transport by small intestine: A status report. Am. J. Physiol. 233(4): E249-254, 1977. - 55 -Speight, J. Acidification of rectal fluid in the locust, Schistocerca Gregaria. M.Sc. Thesis, University of British Columbia, Vancouver, Canada, 1967. Spring, J. Studies on the hormonal regulation of ion resorption in Schistocerca Gregaria. Ph.D. Thesis University of British Columbia, Vancouver, Canada, 1979. Spring, J. and J.E. Phillips. Studies on locust rectum: II. Identification of specific ion transport processes regulated by corpora cardiaca and cyclic-AMP. J. Exp. Biol. 86: 225-236, 1980b. Steele, J.E. and J.H. Tolman. Regulation of water transport in the cockroach rectum by the corpora cardiaca allata system: The requirement for Na +. J. Comp.  Physiol. 138: 357-365, 1980. Tolman J.H. and J.E. Steele. The control of glycogen-metabolism in the cockroach hindgut - The effect of the corpora cardiaca-corpora allata system. Comp.  Bioc. B. 66(1): 59-65, 1980a. Tolman J.H. and J.E. Steele. The effect of the corpora cardiaca-corpora allata system on oxygen consumption in the cockroach rectum - The role of N a + and K +. J. Comp. Physol. 138(4): 347-355, 1980b. Ussing, H.H. D. E r l i j , and U. Lassen. Transport pathways in biological membranes. Annu. Rev. Physiol. 36: 17-49, 1974. Vietinghoff, U., A. Olszewska and L. Janieszewski. Measurements of the bioelectric potentials in the rectum of Locusta Migritoria and Carausius morosus in in Vitro preparations. J. Insect Physiol. 15: 1273-1277, 1969. Wall, B.J. and J.L. Oschman. Structure and function of the rectum in insects. Forschr.  Zool. 23: 193-222, 1975. Warnock, D.G. and J. Eveloff. NaCl entry mechanisms in the luminal membrane of the renal tubule. Am. J. Physiol. 242: F561-574, 1982. Williams, D., J.E. Phillips, W.T. Prince and J. Meredith. The source of short-circuit current across locust rectum. J. Exp. Biol. 77: 107-122, 1978. 

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