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Sodium transport across the locust rectum Black, Kenneth Thomas 1983

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SODIUM TRANSPORT ACROSS THE LOCUST R E C T U M by  KENNETH THOMAS B L A C K 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 F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF ZOOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1983  © K E N B L A C K 1983  In  presenting  requirements  this for  thesis  an  B r i t i s h Columbia,  it  freely  for  available  that  or  understood  that  financial  by  for  his  that  reference  for may  or  be  her  shall  of  Date  DF-fi  (2/79}  the  Library  shall  and  study.  I  copying of by  p u b l i c a t i o n of  not  be  the  make  further this  head It  this  allowed without  Columbia  the  University  representatives.  Z O O L C ^ V  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e Vancouver, Canada V 6 T 1W5  at  granted  permission.  Department  f u l f i l m e n t of  the  extensive  copying or  gain  degree  agree  purposes  department  for  I  permission  scholarly  partial  advanced  of  agree  in  thesis  of  my  is thesis my  written  - ii ABSTRACT  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 of 17.2 mM and a V of -2 -1 1.54 u Eq cm h . The effects of a stimulant (cAMP) and inhibitors (Amiloride and 22 Ouabain) are also investigated. c A M P (1 mM) has no effect on Na flux but does + + cause an increase in short-circuit current (Isc ) (1.28 - 0.12 to 4.80 - 0.28 uEq cm" 2 h"1 ) 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. +  m  m a x  n  +  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 Isc or P.D. Extracts from several neuroendocrine tissues had no conclusive 22 effect on Na fluxes as tested.  - iii T A B L E 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  FIG. 1  DIAGRAM OF USSING CHAMBERS  FIG. 2  I AND UNIDIRECTIONAL C 1 " FLUXES sc  14  FIG. 3  UNIDIRECTIONAL AND NET  16  FIG. 4  FIG. 5 FIG. 6  11 12  3 6  2 2  2 2  Na  FLUXES  +  N a FLUXES OF INDIVIDUAL PREPARATIONS  18  AVERAGE I WITH TIME ACROSS UNSTIMULATED R E C T A sc UNIDIRECTIONAL FLUXES AND I IN RESPONSE TO cAMP  21 23  SC  TABLE 2  2 2  N a FLUXES, I AND P.D. AT VARIOUS ' sc  E X T E R N A L N a CONCENTRATIONS  28  +  FIG. 7  UNIDIRECTIONAL  2 2  Na  +  FLUXES AS A FUNCTION OF  E X T E R N A L N a CONCENTRATION +  AND KINETICS OF  NET N a FLUX  29  +  FIG. 8  HANES-WOOLF PLOT OF N a TRANSPORT KINETICS  FIG. 9  WOOLF-AUGUSTINSSON-HOFSTEE PLOT OF N a  +  TRANSPORT KINETICS FIG. 10  E F F E C T OF OUABAIN ON FORWARD  34 2 2  Na  +  FLUX ACROSS  INDIVIDUAL PREPARATIONS TABLE 3  31  +  E F F E C T OF NEUROENDOCRINE TISSUES ON  37 2 2  Na  +  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 INTRODUCTION  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. from the rectal lumen against large (i.e.  Water is reabsorbed  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 measurements. 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. (1978) and Spring (1979) made measurements of N a , K +  +  Williams et al  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 c A M P stimulation. stimulated by luminal K  This anion transport is also  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 N a - C l 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 Spring (1979) and later Hanrahan (1982) demonstrated that r  the recta but not of N a . +  - 3-  c A M P or corpora cardiaca (CC) extracts cause larqe (3 to lOx) increases in I  and sc  '^Cl  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). Na  +  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 N a - 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 N a - K - A T P a s e , i.e. the "Sodium Pump". Komnick and Achenback (1979) and Ernst et al (1980) have shown, by utilizing (H)-ouabain binding, 3  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 membrance of recta was 127mV, with PD of 64mV, (hemocoel negative) thereby providing a large driving force for N a entry. +  Some of the passive N a 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 2 0 % of N a 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 Na  +  concentration  of 174mM). If all the N a  +  = 22mM at a  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 N a absorption in a similar manner to amino +  acids. Some N a absorption may occur in exchange for cellular H +  is evidence that both these cations are secreted (Hanrahan, 1982; Speight, 1967).  Thus N a  +  or NH^ since there  into the locust rectal lumen  absorption  regulation in terrestrial insects such as the locust.  +  might be implicated in pH  - 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  Hanrahan (1982) ouabain did not penetrate  +  possible that during experiments by  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 C l " 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 N a  +  flux.  Tolman and Steele (1980 a, b), in studies on Periplanta recta,  suggest that there are C C 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 N a also ouabain-sensitive.  is present on the hemocoel. These processes are  +  They suggest that a putative natriferic hormone from the  retrocerebral complex increases N a  +  movement from the hemocoel to N a  +  transport  sites on the lateral membranes. This results in increased of fluid absorption supported by N a  +  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 modified Ussing chambers (Williams et al 1978,  Hanrahan, 1982).  The  two  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. sc  This brief period of open circuit had no visible effect on I  during fluxes, since the I  was identical before and after the open circuit, and was 22  short to significantly alter  sc  too  + 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 N a 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 N a 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 problems because of its low solubility.  special  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. centrifuged  (10,000 g  for 5 min)  and  the  The  resulting mixture  supernatant containing  was  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 J  where:  l-2  =  2  V C  a^TA  -2 -1 ^ * the unidirectional flux (uEq cm h" ) l is the radioactivity of the "hot side" (cpm ml"^) a -1 2 is the increase in radioactivity of the "cold side" (cpm ml ) s  a  C is the concentration of the unlabelled ion in solution 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)  (mM)  - 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  temperature  indicated.  Unless  otherwise  mentioned, the  was 21°C. When temperature was increased in later experiments, this  was achieved using a variable heater, w i t h thermostat.  The experimental p r o t o c o l and the ranges of I  and P.D. considered acceptable  for i n v i t r o r e c t a l preparations were s i m i l a r t o those of Hanrahan (1982) t o permit comparison  of results.  The lower  acceptable  limits  for I  and P.D. were SC  -2 -1 1.0 uEq c m  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 K SO^ 2  Normal Saline  Low Na Saline  High N a Saline  100  Varied  100  5  5  5  10  10  10  +  SO. Free Saline (for Amiloride study)  100  KC1 MgS0  7H O  4  z  Mg(N0 ) 3  2  NaHCO, CaCl  2  10  6H 0  2  2H 0 2  10  10  10  10  5  5  5  5  Na- Methyl SO^  Varied  Choline Chloride  Varied  Amiloride  1  Glucose  10  10  10  10  Sucrose  100  100  100  100  Table Kb) Amino Acids (mM) in all Salines of (a) Alanine  2.91  Asparagine  1.31  Argenine  1.00  Glutamine  5.00  Glycine  11.37  Histidine  1.41  Lysine  1.44  Proline  13.14  Serine  6.50  Tyrosine  1.87  Valine  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  V i a b i l i t y of Unstimulated Preparations and Time to Steady-State  Initial measurements of ^ C l " fluxes were c a r r i e d out across short-circuited r e c t a in normal saline during the 2nd to 4th h a f t e r dissections to determine i f the procedures yielded results s i m i l a r to those previously obtained by Hanrahan (1982) who used i d e n t i c a l methods. For unstimulated preparations, the forward " ^ C l " flux, lumen (L)  to  hemocoel  (H),  2.53 - 0.13 uEq cm'^h"^ -2 0.58 uEq cm  and  back-flux  respectively,  (H  to  yielding  L)  were  an  3.11  average  -  1.21  net  and  flux  of  -1 h  (n=7).  The average I and P.D. during these flux measurements 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 *f"  and H to L fluxes were not significantly different. A l l these values agree closely with previously  published  values  (Hanrahan 1982). Since I transport  for  and P.D.  unstimulated  recta  under  identical  conditions  conformed closely to previous data, these  parameters were subsequently  two  used to assess the v i a b i l i t y of the r e c t a l  preparations, because forward and back fluxes of necessity were measured on different preparations.  The  time course of t y p i c a l I  and " ^ C l " fluxes appears in F i g . 2.  Steady state values were approximated after the first hour.  Transepithelial  22 Na  fluxes were measured in a normal saline (110 mM  across the short-circuited, unstimulated preparations to determine the period for  22  Na to equilibrate w i t h the tissue pool of Na  +  Na  )  required  . A f t e r 30 minutes flux values were  constant (Fig. 3). Therefore, fluxes were subsequently measured between one half and two hours a f t e r the addition of N a , and two to three hours after dissection. F i g . 4 2 2  - 14 -  Fig.  2  -2 -1 The change in I  (uEq cm  h  ) with t i m e a f t e r dissection for t y p i c a l  r e c t a l preparations while monitoring ' ^ C l ~ fluxes. The " ^ C l " fluxes across some individual unstimulated r e c t a l preparations.  ^ C l was  dissection. fluxes.  added to one  side of the chambers  L to H #,0,A,A ; H to L • ,•  V  1 h  after  unidirectional  - 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. N o r m a l saline contained 110 mM  Na . +  Na  dissection in absence of stimulants.  was  added to the saline 4 hs a f t e r  - 18 -  FJg^  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 t i m e for several individual unstimulated preparations, indicating that there was not a great deal of v a r i a b i l i t y with time after the f i r s t flux period f o r any one preparation, compared to that between preparations. flux  ( L to H)  4.68 - 0.65 uEq cm'V  was  and  1  the  Steady-state  back-flux  forward  ( H to L)  was  2.43 - 0.19 uEq c m ~ h . N e t flux was 2.25 uEq c m " h f r o m L to H (n=8). The I ^ sc and P.D. during measurements of N a from L to H were 1.58 - 0.24 uEq cm'^h"^ and 2  •f*  4.9- 1.3 mV.  - 1  Where  1.49 - 0.14 uEq cm'V^  2  22  Na  flux  and P.D.  was  - 1  measured  was 5.1 - 0.6 mV.  H to L, '  the  I sc  was  The s i m i l a r i t y between these  values indicate that preparations used to determine forward flux behaved s i m i l a r l y to those  used t o determine back-flux. These values agreed closely with those reported -2 -1  by Hanrahan (1982; 1.47 uEq c m " h" , indicating that anions hemocoel side, and 5.04 mV, H-side negative.)  were moving to the  A t i m e course of I  and P.D. for a l l  SO  preparations is shown in F i g . 5. Flux Across Stimulated R e c t a  22 The e f f e c t of c A M P on fold increases i n I  g c  Na flux was investigated because this causes several  , P.D., C l " flux and K  +  flux.  conjunction w i t h experiments on the kinetics of N a external N a  +  concentration at 55 mM,  These experiments were done in +  movement discussed below. With  the steady-state forward  flux (L to H) was  1.54 - 0.10 uEq c m ~ h ~ ^ before s t i m u l a t i o n with c A M P and 1.55 - 0.25 uEq cm~ h~^" 2  2  after addition (n=8). Likewise, back flux (H to L) did not change significantly, being 1.31 - 0.11 uEq c m " h  before and 1.35 - 0.09 uEq c m ~ h a f t e r addition of c A M P . 22 The e f f e c t of c A M P on unidirectional N a fluxes across some individual preparations 22 2  _ 1  2  _ 1  is shown in F i g . 6. These results indicate that c A M P has no e f f e c t on was previously reported by Spring et al (1978) using somewhat different  N a fluxes as  - 21 -  Fig. 5  Shows the I with time after dissection across unstimulated r e c t a in sc  +  normal saline containing 110 mM i  sc  •—m,P.D.  o—o  Na  22  and during  Na flux studies.  - 23 -  Fig. 6  22 Unidirectional 1 mM  Na fluxes before and a f t e r the b i l a t e r a l addition of  c A M P to some individual preparations to show the variability  with time and between individual preparations.  c A M P was added at  22 the H  arrow,  VJTJO,*;  45  minutes  HtoL  The response of I  after  the addition  of  + Na :  L to  fluxes. of some individual preparations showing the e f f e c t  of c A M P during experiments in a). 1 m M arrow, 5 h a f t e r dissection.  c A M P was added at the  - 24 -  FIG.6  0  - 25 -  conditions, i.e. his saline contained 55 m M  Na  +  and lacked most amino acids, and he  made no c o r r e c t i o n f o r series resistance of the saline.  I and P.D. showed the sc expected increases after the addition of c A M P , as reported by Spring et al (1978) and Hanrahan  (1982).  I sc  and  P.D.  4.4 - 0.3 mV to 4.80 - 0.28 uEq cm'V  increased  from  1.28 - 0.12 uEq c m " h ^ 2  and 13.2 - 0.8 mV respectively (n=8). Similar  1  increases in I and P.D. were observed at other external N a sc 2).  and  - 1  F i g . 6b shows the time course of I  +  levels and fluxes (Table  in some individual preparations during this  flux studies.  K i n e t i c s of Transepithelial N a Transport Across Short-circuited R e c t a  Some i n i t i a l attempts to investigate N a preparations.  The N a  +  +  transport kinetics were done on unstimulated  concentration was increased by adding sodium  bilaterally f r o m a concentrated stock solution. 2 5 % in I  and P.D. as N a  at a single N a  +  +  This method caused a drop of up to  concentration was increased by 30 mM.  concentration  showed higher  methylsulphate  I  Preparations run  and P.D. than when the same  SC  concentration of N a was achieved by stepwise addition of sodium +  A t 55 m M  the forward  methylsulphate.  2 2 4* 2 1 N a flux (L to H) was 1.54 uEq c m " h" and the back -2 -1  flux  ( H to L)  was  1.31 uEq c m  h  .  1.28 - 0.12 uEq c m ~ h ~ ^ and 4.4 - 0.31 mV 2  The  I  respectively.  and  the  P.D.  were  However, when this N a  +  concentration was attained by adding suitable amounts of N a methyl SO^ to an + 2 1 i n i t i a l l y N a free saline, the I and P.D. dropped f r o m 1.31 t 0.20 uEq c m " h" and +  s  c  - 26 -  of 0.91 - 0.15 uEq cm'V  3.7 - 0.6 mV to an I the N a  +  1  concentration was raised. This drop in I  and 2.1- 0.6 mV respectively a f t e r and P.D. was below the acceptable 22  l i m i t s outlined in the methods. Since c A M P has no e f f e c t s on 110  mM  N a , the kinetics of N a transport were subsequently +  c A M P stimulated preparations. The N a for  +  Na  fluxes at 55 and  determined only f o r  concentration was raised two or three times  each preparation by adding N a methyl-SO^ bilaterally  conditions were overlapped  +  and the experimental  to check variability between runs; e.g. one group of  preparations were run at sequentially increasing N a  +  concentrations of 30, 85, and 140  m M and another group at concentrations of 20, 55, and 120 mM. 2 2 + The  Na  + fluxes at various N a  concentrations appears in Table 2a. The back-  flux was a linear function of e x t e r n a l N a  +  concentration.  The curve for H to L flux  was f i t t e d by linear regression analysis w i t h a resulting c o r r e l a t i o n c o e f f i c i e n t (r) or 0.996. Both the forward and net fluxes showed a non-linear increase (fig. 7) consistent with Michaelis-Menten  kinetics.  variations of the Michaelis-Menten  These values of net N a  +  equation:  V S _ max ~ S+ K m V = J ^ max max V  where:  3  = N a concentration max K  m  = Na  ,Na concentration at k maximum J max  flux were confirmed using  - 27 Table 2(a) 22  Na fluxes at various external Na concentrations under short-circuited conditions (x - SE, n = 6-8) N a Fluxes (uEq.cm" h"l) (H to L) 2 2  Na  Concentration (mM)  (L to H)  +  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 +  I (uEq.cm sc  -2 -1 h~ )  P.D. (mV)  (mM) 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 -  Fig,?  22 Unidirectional  Na  fluxes as a function of external N a concentration  under s h o r t - c i r c u i t conditions at 20° C. Forward flux L to H Back flux H to L  .  Line for H to L flux was f i t t e d by  linear regression analysis with a corelation c o e f f i c i e n t of 0.996. The  curve showing  kinetics of net N a  +  flux w i t h  changing N a  +  concentration, is the difference between mean unidirectional fluxes in a).  -29  FIG.  1  20  1  1  40  EXTERNAL N a  CO +  i 80  CONCENTRATION  1  100 (mM)  1  120  7  1—  140  - 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 the line. With this plot, the K  was 17.14 mM with a V of 1.54 uEq cm m max  -2 -1 h . The  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. 3  The K  m  was 17.2 mM  and the V  max  was  -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 1 mM  Na fluxes were measured before and 1 h after the addition of  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  22 divided by the net  Na  flux against the N a  +  concentration. The line was  f i t t e d using linear regression analysis giving the correlation c o e f f i c i e n t of 2 0.991. The V the K  m  max  is equal to the slope of the line, 1.54 uEq cm  equals the ^  m  a  x  multiplied by the y-intercept, i.e. 17.14  1 h  mM.  , and  - 32 -  FIG. 8  100  80-1  10  ?0  30  40  50  60  Na* CONCENTRATION  70  (mM)  80  9*0  - 33 -  22 A Woolf-Augustinsson-Hofstee plot of the net 22+ + Na  flux divided by the external Na  + Na  concentration.  flux against the net The line was  fitted  by linear regression analysis with a resulting c o r r e l a t i o n c o e f f i c i e n t of -0.951. V"  m a x  The K  is equal to the negative of the slope, 17.2 mM -2 -1 is equal to the y-intercept, 1.54 uEq cm h .  and  the  - 35 -  In a separate series of experiements the inhibition caused by amiloride was found to be reversible. The forward  22  + 2 N a flux increased f r o m 3.35- 0.55 uEq c m  h  1  in the  presence of amiloride t o 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.  A m i l o r i d e had no e f f e c t on I or P.D. sc  Before addition of amiloride t o c A M P  ry "1 stimulated r e c t a , I was 10.25 - 0.87 uEq c m h and the P.D. was 18.4 - 1.8 mV. ' sc ^ One hour a f t e r the addition of amiloride, the I and P.D. were respectively 10.61 ± 0.87 uEq c m " h" and 18.9 - 1.9 mV. In the second set of experiments when the  preparations  7.96 - 0.82 uEq c m  were  initially  bathed  in  amiloride,  the  I  was  2 1 h and the P.D.  was 18.7 - 1.4 mV. A f t e r the amiloride was 2 1 removed, the I was 8.11 - 0.77 uEq c m " h" and the P.D. was 18.5 - 1.1 mV for the sc ^ •4*  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  a f t e r stimulation, is not coupled t o N a transport.  A m i l o r i d e does  appear t o inhibit transepithelial N a movement, but the mechanism of inhibition is unclear.  22 The e f f e c t s of ouabain on  + Na  flux were not so conclusive. The preparations  were not stimulated with c A M P and were bathed with a saline containing 110 mM The  22  Na fluxes before the addition of 1 m M  (L t o H) and 2.39 - 0.18 uEq c m " h " 2  1  Na . +  2 1 ouabain were 3.85 - 9.75 uEq c m " h~  ( H to L) (n=8). Sixty minutes a f t e r the addition  + 2 1 of ouabain the forward flux had dropped somewhat t o 3.53 0.68 uEq c m " h" , whereas 2 1 the backflux (H t o L) stayed the same at 2.36 - 0.21 uEq cm" h" . However, this  - 36 -  average drop i n the influx (L to H) is not significant.  In all but one run, the N a  +  forward influx was slightly lower a f t e r 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 a f t e r the addition of ouabain in agreement with  earlier observations by Hanrahan (1982). Before the addition of I m M ouabain to the + 2 X unstimulated preparations, the I was 1.77 - 0.19 uEq c m " h" and the P.D. was + * + 2 1 5.2 - 0.4 mV, and a f t e r addition of this inhibitor the I was 1.79 - 0.13 uEq c m " h~ ' sc ^ and the P.D. was 5.1 - 0.5 mV.  A recent review by Anstee and Bowler (1979) indicates that N a transport in most insect epithelia is unusally insensitive to ouabain.  These workers have shown that  ouabain inhibition is very temperature-dependent.  Temperatures  w e l l above room  temperatures are often necessary to observe an e f f e c t with ouabain. Indeed ouabain inhibition of N a - K - A T P a s e isolated from locust rectum is neglible at room ture and only 5 0 % at 32° (Donkin and Anstee 1980).  tempera-  Moreover, the c u t i c l e and basal  tissue layers on the mucosal and serosal sides respectively of locust r e c t a l epithelium may act as diffusion barriers, thereby slowing the access of ouabain t o the basolateral c e l l border, where N a - K - A T P a s e has been located in r e c t a of dragonfly larvae (Komnick  et a l 1979).  To overcome this diffusion problem, preparations were  stimulated w i t h c A M P and exposed t o 1 mM  ouabain for longer periods (3 h), w i t h the  anticipation that this should give ouabain ample time t o 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  r e c t a l preparations, before Ouabain.  1 mM  of 1 m M  and a f t e r the b i l a t e r a l addition  ouabain in normal saline lacking c A M P was added to the  preparations at the arrow. temperature was 21° C .  The saline contained 110 m M N a  +  and the  - 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 As a compromise, the temperature was reduced to 29-30° C.  and  A t this temperature the  preparations appeared viable for at least six t o seven hours as judged by stable I P.D.  Donkin and  Anstee (1980) report 4 5 %  P.D.  inhibition of isolated  locust  and rectal  22 N a - K - A T P a s e at this temperature. I found the f o r w a r d 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 c m ~ h " ^ a f t e r its addition (n=6). This was a 36.5% decrease in active 2  22 N a influx (L t o H) and is s t a t i s t i c a l l y significant by a standard t test (©<=0.05). Again I  and P.D. did not change significantly a f t e r the addition of ouabain. The I 2  and P.D. were 11.96- 0.38 uEq cm" 2 11.423 0.95 uEq cm" After  a  3  h  hi  1  1 h"  and 17.5 - 0.5 mV  before adding ouabain and  1  exposure  10.85 - 1.52 uEq cm'V  g c  and 18.5 - 1.5 mV to  1  mM  at one hour after adding this inhibitor.  ouabain  at  30°  and the P.D. was 17.5- 2.5 mV.  P.D.  is similar to the normal decay of I and P.D. ' sc ouabain. In conclusion, a t , 30° C  ouabain causes p a r t i a l  C.  the  I  was  still  This slight decline in I and sc with t i m e in the absence of  inhibition of N a  transport  approaching the reduction predicted f r o m studies with the isolated N a - K - A T P a s e from this tissue.  Again lack of e f f e c t of ouabain on I  and P D c o n f i r m the conclusion by  Hanrahan (1982) that C l " transport (i.e. I ) is independent of N a transport.  - 40 -  E f f e c t s of E x t r a c t s f r o m Neuroendocrine Tissues on N a  Since insect hormones which influence body N a  +  +  Absorption by Locust R e c t a  levels and epithelial transport  have not been reported, except f o r some preliminary and indirect evidence by Steele et al (1980), the e f f e c t s of e x t r a c t s f r o m the major locust neurosecretory tissues on rectal  22  Na  +  flux was also investigated.  22  Na  +  influx was selected because i t includes  both a c t i v e and passive components so that a change in either could be detected, and then the s p e c i f i c component i d e n t i f i e d later i f necessary. A n i n i t i a l test was run using extracts f r o m the brain, the sub-esophageal ganglion, pars intercerebralis,the thoracic and  abdominal ganglia.  preparations  The results appear i n Table 3.  Short-circuited r e c t a l  were bathed in normal saline containing 110 m M N a  at 2 1 C. u  The  22 Na  influxes were measured both before and a f t e r measuring dose response curves  of I  to the e x t r a c t (by J. Proux unpublished observation), so that maximum amounts 22  of gland e x t r a c t s were present during the second  Na flux measurement.  -41 Table 3(a) The Effect of Neuroendocrine Tissues on T s l a Influx Across Short-circuited Locust Recta Z  Gland A c c u m m u l a t e d Dose (8.2 gland-equivalents/5ml)  +  Flux (L t o H)(uEq.cm~ h~ ) 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 G a n g l i a 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%  2.49 ±0.23  2.38 ±0.24  -4%  Thoracic Ganglia 1-3 (n=3) 110 mM  Na  +  * significant atc<*0.05 Table 3(b) Before I sc 6.0  Brain  + +  After PD  'sc  PD  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  +  0.4  3.6 ± 0.3  23.5 ± 1.6  14.5 ± 1.5  +  0.5  6.0 ± 1.0  5.0 ± 0.9  8.0 ± 0.8  +  0.6  4.1 ± 0.6  8.0 ± 1.2  8.4 ± 1.1  +  0.7  9.5 ± 2.1  7.7 ± 1.7  6.4  Thoracic Ganglia 1-3 Abdominal Ganglia 4-7  3.5  Abdominal Ganglia 8  4.0  Thoracic Ganglia 1  6.3  Thoracic Ganglia 2  7.3 8.3  Thoracic Ganglia 3 T G 1-3, 3 Glands 1 1 0 m M N a T G 1-3, 3 Glands 2 0 m M N a  +  +  6.7 8.5  + + + +  6.2 ± 1.1  0.8  7.0 ± 0.6  10.3 ± 1.2  8.0 ± 1.5  0.9  10.3 ± 1.8  15.7 ± 1.8  19.7 ± 1.8  0.4  3.8 ± 0.4  13.2 ± 1.1  7.2 ± 0.9  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 e f f e c t i n i t i a l l y , that of increasing in  Na  influx by 7 5 % .  +  Therefore, the e f f e c t of this neuroendocrine tissue was studied in more depth.  These further tests were done with a pooled e x t r a c t of thoracic ganglia 1, 2, and 3.  The equivalent of three ganglia were added to the hemocoel side of the r e c t a  bathed with 5 ml of saline containing 20 mM  Na . +  A lower Na concentration was used  in case a n a t r i f e r i c f a c t o r might change K  rather than V of a c t i v e N a transport. m max fluxes before and a f t e r the addition of e x t r a c t were 0.87 - 0.12 and 3  The forward Na  r  + 2 1 + 0.85 - 0.13 uEq cm" h respectively (n=8). This result shows that at a N a concent r a t i o n near the K for N a net flux, which is also a t y p i c a l N a value observed in the m 22 + r e c t a l lumen in situ, e x t r a c t s of t h o r a c t i c ganglia had no e f f e c t on the Na influx. +  +  The same experiment was repeated w i t h a saline containing 110 mM 22 there was no significant change in the forward thoracic gland e x t r a c t the N a 2 2  +  +  Again  + Na  flux.  Before the addition of  flux (L to H) was 2.49 - 0.23 uEq c m " h  + 2 1 it was 2.38 - 0.24 uEq cm" h~ (n=6).  Na .  2  _ 1  and a f t e r  These more extensive results suggest that 22 +  extracts of t h o r a c t i c ganglia have no e f f e c t on  Na  influx, i n contradiction t o  results in a few preliminary experiments. The I  and P.D. values during experiments with e x t r a c t s f r o m neuroendocrine  tissues appear in Table 4b.  - 43 -  DISCUSSION  The  purpose of this study was t o c h a r a c t e r i z e sodium absorption under appro-  priate in vitro conditions, similar to those employed by Hanrahan (1982) in his study of KC1 absorption across locust r e c t a . He concluded that electrogenic C l " transport was the main a c t i v e process in this tissue a f t e r stimulation with C T S H (Chloride Transport Stimulating Hormone) or c A M P , which is the second messenger of C T S H . enhanced anion absorption is coupled  to passive absorption of K . +  extensive evidence that C l " transport was not coupled t o N a  +  He  This  provided  absorption or H C O ^  secretion (Hanrahan and Phillips, 1983a) as i t is in vertebrate epithelia ( F r i z z e l l et a l 1979).  In this study additional evidence for the lack of Na-coupled C l " transport in  locust r e c t a was provided.  There was considerable evidence for a c t i v e 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 f r o m the lumen of ligated r e c t a 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). external K  +  In the absence of  and C l " , a c t i v e N a absorption, even f r o m dilute solutions, sustained fluid +  transport across everted r e c t a l sacs at low rates, against N a concentration d i f f e r e n +  ces and in the absence of a sizeable T E P (Phillips et al 1982). These results suggested a low K  t  for N a  across  everted  +  transport. Moreover, I m M ouabain inhibits f l u i d transport by 5 0 % rectal  sacs  (Irvine  and  Phillips,  1970) although  incubation  -44 -  conditions were clearly sub-optimal  i n these early experiments (see Phillips 1982).  Both Williams et al (1978) and Spring and Phillips (1980b) measured a net flux of  2 2  Na  -1  +  -2  f r o m lumen to hemocoel across short-circuited locust r e c t a (4.4 and 2 u Eq h" c m " and w i t h flux ratios of 4:1 and 2:1 respectfully).  However, these authors did not  correct f o r the resistance of the saline bathing  the r e c t a and this saline was  physiologically less satisfactory than that used by Hanrahan (1982). measurements were also made at a single high e x t e r n a l N a concentration. +  There In this  present study, c o r r e c t i n g for saline resistance and using Hanrahan's saline, I obtained a somewhat lower J uEq h ^  -1-2 cm  max  for net  22  Na  +  flux under short-circuited conditions (about 1.5  over the 2nd t o 4th h).  This is about 1 0 % of J  max  for active C l  absorption under the same conditions ( l O m M e x t e r n a l K ; Hanrahan and Phillips, +  1983a).  Results i n this study c o n f i r m a report by Spring and Phillips (1980b) that 22  c A M P and C T S H did not a f f e c t  + Na  fluxes under 'near' short-circuited conditions.  It is now w e l l established for most absorptive epithelia, especially in vertebrates, that sodium enters passively down a favourable e l e c t r o c h e m i c a l gradient due t o a low intracellular level of N a  +  and a favourable P.D. across the mucosal membrane.  situation is maintained by extrusion of N a against high N a  +  +  This  from the c e l l across the serosal membrane  concentration and P.D. due t o N a - K - A T P a s e . +  evidence for such a mechanism in locust rectum.  There is indirect  A s in many other epithelia, the  r e c t a l c e l l interior of locusts and other insects is negative t o the outside (Phillips, 1964; Vietinghoff et a l 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 r e c t a (Phillips, 1964,  1980)  and this has also been confirmed by microprobe  analysis f o r C a l l i p h o r a r e c t a l pads (Gupta e t a l 1980).  Hanrahan e t al (1982) used  double-barrelled ion-sensitive microelectrodes t o measure intracellular N a  +  activities  and e l e c t r i c a l potential. He was thus able t o c a l c u l a t e electrochemical  differences  across a p i c a l and basolateral c e l l borders of locust r e c t a exposed t o varying Na  +  a c t i v i t i e s and with transepithelial P.D. clamped at OmV.  remained between 3 and 15mM electrochemical ImM.  gradient  under a l l conditions  for passive N a  Intracellular N a levels +  and there was a favourable  entry when luminal N a  +  luminal  +  levels exceeded  In Hanrahan's saline (i.e. H O m M N a ) the net electrochemical +  difference  favouring passive entry (mucosal) and active e x i t (serosal) was very large (127mV). Hanrahan noticed no e f f e c t of I m M ouabain on these gradients at room temperature unpublished  research).  (This  apparent  observations made i n this study.) accumulation of K  inconsistency  c a n now  be explained  Finally, Hanrahan (1982) demonstrated  by  active  inside r e c t a l epithelial c e l l s t o a control l e v e l of 70mM when  +  K-depleted r e c t a were re-exposed to l O m M K  +  on the hemocoel side.  This is again  consistent w i t h a N a - K exchange pump on the serosal membrane. +  A  ouabain  +  sensitive (kC 10~^M) N a - K - A T P a s e  with  biochemical  properties  similar t o those observed in other tissues has been found in r e c t a of locusts and other insects (Peacock, 1977, 1981;  K o m n i c k and Achenbach, 1979).  ATPase f r o m locust r e c t u m , lOOmM activity.  Na  +  and 20mM K  +  In the case of the  are needed f o r m a x i m a l  K o m n i c k and Achenbach (1979) used ^(H)-ouabain and autoradiography to  localize the N a - K - A T P a s e at the basolateral c e l l border of r e c t a l epithelial cells in  -46 -  dragonfly larvae, but this location has not been re-investigated for t e r r e s t r i a l insects. Donkin and Anstee (1980) argue that the failure t o demonstrate ouabain inhibition of Na -dependent transport processes i n many insect epithelia was because experiments +  were conducted at room  temperature.  They showed that ouabain inhibition of  N a - K - A T P a s e f r o m locust is neglible at room temperature, is only 4 5 % at 30°C, and is similar to that of vertebrate N a - K - A T P a s e at 37°C. In the present study, I found that 22 ouabain only inhibited a c t i v e influx of  + Na  29°C, at which point the inhibition of N a  i f the room temperature were raised to flux was similar to that of N a - K - A T P a s e  +  22 reported by Donkin and Anstee (1980) (i.e. 3 6 % ) .  When  + Na  fluxes were inhibited by  ouabain there  was no e f f e c t on Cl~-dependent I which reconfirms Hanrahan's sc conclusion that electrogenic C l " transport in locusts is not coupled to N a transport. r  +  This idea is f u r t h e r supported by the experiments w i t h amiloride. This agent inhibits 22 active net  + Na  flux without a f f e c t i n g Cl"-dependent I . F i n a l l y , varying luminal SC  N a levels in the present study did not change Cl-dependent I  across s t i m u l a t e d r e c t a  during k i n e t i c studies. 22 Demonstrations of p a r t i a l inhibition of net  + Na  flux across locust r e c t u m by  ouabain and amiloride provide additional evidence f o r a mechanism of N a similar to that found i n vertebrates.  +  transport  N a C l co-transport generally occurs in "leaky"  vertebrate epithelia where the transepithelial P.D. is too small to drive C l  absorption  by e l e c t r i c a l coupling, which occurs i n tight epithelia ( F r i z z e l l et al 1979).  Since  locust r e c t u m is e f f e c t i v e l y tight (Hanrahan, 1982) i t is not surprising that anion and c a t i o n 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.  A m i l o r i d e inhibits N a  entry into other epithelia via selective channels (e.g. frog  +  skin, Benos et a l 1968; rabbit colon, F r i z z e l l and Turnheim, 1978) or by f a c i l i t a t e d exchange f o r H  +  (Liedtke and Hopfer, 1977).  The dose-response relationship f o r  amiloride inhibition differs by two-to-three orders of magnitude for these two entry processes (Warnock, et a l 1982).  Only a high dosage of amiloride was used i n the  present study, so i t is not possible to distinguish between the two types of entry processes which may be inhibited i n 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 I m M had no significant e f f e c t on the mucosal P.D., indicating a very low conductive permeability of this membrane t o N a (P^a- -)' The mucosal P.D. +  was largley a K  +  1  diffusion potential (55mV change per decade change in luminal K ) +  with a smaller electrogenic component associated with the a p i c a l C l " pumps (about lOmV). In support of low P ^  g  of the a p i c a l 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 t o the lumen side when locust r e c t a l sacs were bathed b i l a t e r a l l y in an  isosmotic sucrose solutions. Thus N a occurs  largely by coupled  +  entry into this tissue from the lumen probably  mechanisms.  A c t i v e absorption of neutral amino acids  (Balshin, 1973, Balshin and Phillips, 1971), acetate  (Baumeister et a l 1981) and  phosphate (Andrusiak, 1974) have been shown in locust rectum. In the case of glycine  - 48 -  the transport process is c l e a r l y coupled t o N a 1973).  +  (Balshin and Phillips, 1971; Balshin,  The lumen side is a c i d i f i e d by an active mechanism i n situ (Speight, 1967,  Phillips,  1964) and across  Communication).  unstimulated  The magnitude of this H  this occurs by N a - H +  +  in vitro  (Thompson,  Personal  secretion is 0.4 to 1.4 uEq f T ^ c m " . If 2  exchange at the a p i c a l border, i t would account f o r a large  +  portion o f the net N a  recta  +  entry into r e c t a l epithelial c e l l s of the locust. Future work  might be to investigate the H  +  secretion and the e f f e c t s of amiloride on H  to discover i f i t is comparable t o N a  +  +  movement  inhibition observed as a result of this drug. It  might also prove interesting to discover i f amiloride has any e f f e c t s on amino acid movement thereby confirming the coupling of N a transport to amino acid transport. +  The low «  for net N a - a b s o r p t i o n in the locust r e c t u m (17mM F i g . 8 and 9) is +  t  similar t o values for other vertebrate epithelia. The K^. for N a - a b s o r p t i o n is 22mM in +  toad urinary bladder (Ussing et al 1974), l O m M 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 e p i t h e l i a were short-circuited. These tissues are a l l "tight" e p i t h e l i a ; i.e. only a small percentage of ion flow occurs v i a a paracellular pathway.  These "tight" vertebrate  e p i t h e l i a a l l exhibit t r a n s e p i t h e l i a l resistances  2 greater  than 10,000 A  cm  (Lewis and Diamond, 1976).  When stimulated 2  with  c A M P , locust r e c t a have lower trancellular resistances (50-150 It c m ) but only 5 % of current  flow  is by the paracellular route (Hanrahan, 1982).  Another  difference  between locust r e c t a l e p i t h e l i a and vertebrate e p i t h e l i a is that N a - s u b s t i t u t i o n has a dramatic e f f e c t on I across these vertebrate e p i t h e l i a (Lewis and Diamond, 1976) but sc not locust rectum. +  -49 -  It has been shown that c A M P and C T S H did not a f f e c t N a fluxes even though I sc and ^ C l ~ fluxes increased (Spring and Phillips, 1980b). These authors only tested the +  3  actions of other neuroendocrine tissues from locusts on I and not on N a sc these endocrine e x t r a c t s acted on a N a  +  absorption  their e f f e c t would not have been detected.  +  fluxes. If  process which is electroneutral,  Tolman and Steele (1980a, b) have  provided some indirect evidence for a n a t r i f e r i c f a c t o r in C C . of cockroaches, which enhances f l u i d reabsorption  by r e c t a l sacs.  P r i o r to this study there were no  systematic surveys of insect endocrine organs for factors d i r e c t l y influencing N a reabsorption  across insect r e c t a or indeed any aspect of N a  +  regulation i n insects.  Using procedures s i m i l a r to those f o r e x t r a c t i o n of several water-soluble neuropeptide hormones, I was unable to detect absorption,  although thoracic  further study (Table 2). vasopressin-like hormone.  ganglia  +  insect  any f a c t o r influencing r e c t a l N a  +  gave mixed results which perhaps warrant  It was in the t h o r a c i c ganglia that Proux (1981) isolated a It is s t i l l possible that a locust n a t r i f e r i c factor exists  which is either very unstable or is not e x t r a c t e d by the procedures used for hydrophilic polypeptide hormones. This possibility should be investigated.  SUMMARY  1.  The k i n e t i c s for N a  flux across the unstimulated locust r e c t u m are established  with the K m being about 17.2 m M and V 3  2.  of 1.54 uEq cm m a y  -2 -1 h .  ^  It is v e r i f i e d that c A M P has no e f f e c t on the N a flux across r e c t a . +  - 50 -  A m i l o r i d e is found t o inhibit  Na flux from lumen t o hemocoel by 3 3 % and net  Na absorption by 7 5 % . The e f f e c t of amiloride is reversible.  22 Ouabain (1 mM) has no e f f e c t on  Na flux at room temperature (21°C) but at  higher temperatures (29°C) there is a 3 7 % inhibition of N a influx from lumen t o hemocoel. Extracts  f r o m several neuroendocrine tissues had no conclusive e f f e c t s on the  fluxes of N a  +  across the locust rectum.  - 51 BIBLIOGRAPHY  Andrusiak, E.W. Resorption of Phosphate, C a l c i u m , and Magnesium in the in V i t r o Locust R e c t u m . M.Sc. Thesis, University of B r i t i s h 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 A c i d s in V i t r o by the R e c t u m of the Desert Locust Schistocerca Gregaria. Ph.D. Thesis, University of B r i t i s h Columbia, Vancouver, Canada, 1973. Balshin, M. and J.E. Phillips. A c t i v e Absorption of amino acids in the r e c t u m of the desert locust (Schistocerca Gregaria). Nature London 233: 53-55, 1971. Baumeister, T., J. Meredith, W. Julien, and J. Phillips. A c e t a t e transport by locust r e c t u m 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. A m i l o r d i e : 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 N a concentration on N a transport across frog skin. J. Gen. Physiol. 47: 879, 1964. Chamberlin, M. Metabolic Studies in Locust Rectum. Ph.D. Thesis University of B r i t i s h 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 e f f e c t of temperature on the ouabain-sensitivity of N a - K - a c t i v a t e d +  +  ATPase and f l u i d 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 J r . Relationship between l o c a l i z a t i o n of Na -K +  +  ATPase, cellular fine structure, and reabsorptive and secretory e l e c t r o l y t e  transport in Current Topics in Membrance and Transport (F. Browner, A. K l e i n z e l l e r and E.L. Boulpaep, eds.) A c a d e m i c Press, London. Vol. 13 pp. 355-385, 1980. F r i z z e l l , R.A., M. F i e l d 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. U n i d i r e c t i o n a l sodium influx and the e f f e c t s of Amphotericin B and A m i l o r i d e . J. Mem.  B i o l . 40: 193-211,  1978.  Gupta, B.L., B.J. Wall, J.L. Oschman and T.A. H a l l . D i r e c t microprobe evidence of local concentration gradients and r e c y c l i n g of electrolytes during fluid reabsorption in the r e c t a l papillae of C a l l i p h o r a . J. exp. B i o l . 88: 21-47, 1980. Hanrahan, J.W. Hormonal regulation of chloride in locusts. The Physiologist 21: 50, 1978. Hanrahan, J.W. C e l l u l a r mechanisms and regulation of KC1 transport across an insect epithelium. Ph.D. Thesis, University of B r i t i s h Columbia, Vancouver, Canada, 1982.  - 53 Hanrahan, John and J.E. Phillips. Mechanisms and c o n t r o l of salt absorption in locust rectum. Am.  J. Physiol. 244: R131-142, 1983  Hanrahan, J.W. and J.E. Phillips. C e l l u l a r mechanism of KC1 absorption in insect hindgut. J. Exp. B i o l . In Press. 1983b.  Irvine, H.B. and J.E. Phillips. E f f e c t s of respiratory inhibitors and ouabain on water transport by isolated locust r e c t u m J. Insect Physiol. 17: 381-393, 1970.  Komnick, H. and U. Achenbach. Comparitive biochemical histochemical and autoradiographic studies of N a / K - A T P a s e in the r e c t u m of dragonfly larvae (odonata, aeshnidae). +  +  Eur. J. C e l l B i o l . 20: 92-100, 1979.  Leaf, A. Transepithelial Transport and its hormonal c o n t r o l 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, Liedtke, C M .  1976.  and U. Hopf er. Anion transport in brush border membranes isolated  f r o m r a t small intestine. Biochem. Biophys. Res. Commun. 76: 579-585, 1977. Maddrell, S.H.P. Insect Malpighian tubules. In Transport of Ions and Water in A n i m a l s (B.L. Gupta, R.B. Moreton, J.L. Oschman, B.J. Wall, eds.), pp 541-570 A c a d e m i c Press, London, 1977. Maddrell, S.H.P. C h a r a c t e r i s t i c s of epithelial transport in insect Malpighian tubules. In Current Topics in Membranes and Transport (F. Bronner and A. K l e i n z e l l e r , eds.) A c a d e m i c Press, London, Vol. 14 pp 427-463, 1980.  Peacock, A.J. Distribution of N a - K +  +  a c t i v a t e d A T P a s e in the hindgut of two insects,  Schistocerca and Blaberus. Insect B i o l . 7: 393-395, 1977.  - 54 Peacock, A.J. Further studies of the properties of locust r e c t u m N a - K +  +  ATPase  with p a r t i c u l a r reference to the ouabain sensitivity of the enzyme. Comp. Bioc. Physiol. 68C: 29-34, 1981.  Phillips, J.E. R e c t a l absorption in the desert locust, Schistocerca Greqaria Forskal II. Sodium, potassium and chloride. J. Exp. Biol. 41: 39-67, 1964. Phillips, J.E. E x c r e t i o n in insects: function of gut and r e c t u m 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. S.G.  Schultz, eds.) pp 333-353 A c a d e m i c Press, New  Kleinzeller,  York. 1977b.  Phillips, J.E. E p i t h e l i a l transport and control in r e c t a of t e r r e s t r i a l insects. In Insect Biology in the Future (M.L. L o c k e 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 c o n t r o l of salt and water balance: E x c r e t i o n . In Endocrinology of Insects (H. Lauder and R. Downer eds.) Alan R. Liss, New  York. 1983.  Proux, J . Regulation neuroendocrine de l a diurese chez le criquet migrateur. D o c t o r a l 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  c h a r a c t e r i s t i c s 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 s m a l l intestine: A status report. Am.  J . Physiol. 233(4): E249-254, 1977.  - 55 Speight, J . A c i d i f i c a t i o n of r e c t a l 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 U n i v e r s i t y 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 c a r d i a c a and c y c l i c - A M P . J. Exp. B i o l . 86: 225-236, 1980b.  Steele, J.E. and J.H. Tolman. Regulation of water transport in the cockroach r e c t u m by the corpora cardiaca allata system: The requirement for N a . J. Comp. +  Physiol. 138: 357-365, 1980. Tolman J.H. and J.E. Steele. The c o n t r o l of glycogen-metabolism in the cockroach hindgut - The e f f e c t of the corpora cardiaca-corpora allata system. Comp. Bioc. B. 66(1): 59-65, 1980a. Tolman J.H. and J.E. Steele. The e f f e c t of the corpora cardiaca-corpora allata system on oxygen consumption in the cockroach r e c t u m - 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 b i o e l e c t r i c potentials in the r e c t u m of Locusta M i g r i t o r i a and Carausius morosus in in V i t r o preparations. J. Insect Physiol. 15: 1273-1277, 1969. Wall, B.J. and J.L. Oschman. Structure and function of the r e c t u m in insects. Forschr. Zool. 23: 193-222, 1975. Warnock, D.G. and J. E v e l o f f . N a C l entry mechanisms in the luminal membrane of the renal tubule. Am. J. Physiol. 242: F561-574, 1982. Williams, D., J.E. P h i l l i p s , W.T. P r i n c e and J. Meredith. The source of short-circuit current across locust rectum. J. Exp. B i o l . 77: 107-122, 1978.  


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