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Resorption of phosphate, calcium and magnesium in the in vitro locust rectum Andrusiak, Edward William 1974

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RESORPTION OF PHOSPHATE, CALCIUM, AND MAGNESIUM IN THE IN VITRO LOCUST RECTUM  by  EDWARD WILLIAM ANDRUSIAK B . S c , U n i v e r s i t y of Manitoba, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January, 1974  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfilment of  an advanced degree at the U n i v e r s i t y of the L i b r a r y I  further  for  this  freely  available  for  agree 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  scholarly  by h i s of  s h a l l make it  British  the  requirements  Columbia,  I agree  reference and copying o f  this  for  that  study. thesis  purposes may be granted by the Head of my Department or  representatives. thesis for  It  financial  is understood that gain shall  written permission.  Department of The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  7  not  copying or  publication  be allowed without my  ABSTRACT  The a b i l i t y of the l o c u s t rectum i n v i t r o to resorb C a , + +  Mg  ++  , and PO^ was studied.  The rectum has a low permeability to Ca  ++  ,  ++ ++ but Ca i s not accumulated against concentration d i f f e r e n c e s . Mg was not accumulated i n the basal compartment of the r e c t a l sac even when M g  ++  concentration gradients were favourable f o r net d i f f u s i o n .  Phosphate was found to be accumulated by the rectum against a threef o l d concentration d i f f e r e n c e . The r e c t a l t i s s u e incorporated inorganic phosphate i n t o organic forms, but phosphorus t r a n s f e r r e d into the basal compartment was found to be i n the form of inorganic phosphate.  Uptake into the  basal compartment can be described by Michaelis-Menton s a t u r a t i o n kinetics.  The r e s o r p t i o n of water by the rectum d i d not increase (by  solute drag) the amount of PO^ accumulated i n the basal compartment, except a t very high PO^ concentrations i n the a p i c a l bathing medium. Metabolic poisons such as KCN and IAA i n h i b i t e d PO^ accumulation i n the basal compartment but d i d not i n h i b i t PO^ entry into the t i s s u e from the a p i c a l bathing medium.  Arsenate, a competitive i n h i b i t o r of PO^  uptake i n other systems, i n h i b i t e d PO^ entry into the t i s s u e . A mechanism f o r PCL uptake i n t o the t i s s u e i s proposed.  1 i 1  TABLE OF CONTENTS Page INTRODUCTION  1  MATERIALS AMD METHODS  6  Animals  6  Preparation of the Everted Rectal Sacs  6  Incubation Procedure  7  Composition of Bathing Media  8  Measurement of Ion Concentrations i n Media and Tissues  11  Electropotential Differences Across the Rectal Sac . . . . .  12  Treatment of Results  14  RESULTS  16  V i a b i l i t y of the In Vitro Rectal Preparation  16  Test for Accumulation of Ca , Mg  17  Net Ca Net Mg  ++  , and PO^  Movement  1"  Accumulation  19  Net PO^ Accumulation  22  Tissue Contribution to Uptake of PO. in the Basal Compartment Type of PO^ (Inorganic and/or Organic) i n the Basal Compartment  25 28  Effect of Water Uptake on PO. Accumulation in the Basal Compartment  29  Kinetics  29  Page I n h i b i t i o n of PO^ Transfer Further C h a r a c t e r i z a t i o n of the System DISCUSSION SUMMARY  .....  3 2  34 3 8  4 6  V  LIST OF TABLES Table I. II.  ;  10  Composition of Bathing Media The Net Movement of C a i n t o the Rectal Tissue and i n t o the Basal Bathing Medium, Over 3 Hours of Incubation i n Media Containing Various Amounts of C a + +  + +  III. IV.  Page  Net P Uptake i n E i t h e r Control or Poisoned Tissue, A f t e r 6 Hours of Incubation  2 1  3 2  Various Fractions of PO4, Determined by Chemical Analysis and Radiotracer Estimation ( P ) , i n InCubated and Unincubated Recta  .  33  3 2  35  vi  LIST OF FIGURES Figure  Page  1.  Diagram o f experimental absorption iji v i t r o  2.  Apparatus used f o r measurement of trans r e c t a l potentials  3.  4.  set-up to study r e c t a l  Trans r e c t a l potential d i f f e r e n c e s (apical side p o s i t i v e ) across everted r e c t a l sacs with time a f t e r preparation The net rate of water movement across the r e c t a l wall i n the absence and presence of an osmotic gradient  ~ 1 3  15  ^  The net movement o f C a and water across the r e c t a l w a l l , a f t e r 3 hours of incubation i n Ringer containing various amounts o f C a ..  20  The net movement of M g and l-LO across the r e c t a l wall at various M g concentrations i n the apical bathing medium, with and without net water movement across the r e c t a l wall .  23  The t o t a l amount o f M g i n r e c t a l t i s s u e a f t e r 4 hours of incubation, with and without sucrose present i n the a p i c a l bathing medium  24  The basal: apical (B/A) concentration r a t i o o f P0^ across i n v i t r o r e c t a l sacs with time  26  The f i n a l P 0 ^ r a t i o (B/A) and the net movement o f water across the r e c t a l sacs ( a f t e r 6 hour o f i n cubation) when the P 0 * concentration o f the bathing media was varied  27  The net rate o f P O 4 appearance i n the basal compartment and the net water movement across the r e c t a l sac f o r a range of P O 4 concentrations i n the apical bathing medium  30  Double reciprocal p l o t of the net rate o f P O 4 entry into the basal compartment as a f u n c t i o n of the P O 4 concentration i n the apical bathing medium  31  + +  + +  6.  ++  ++  7.  8. 9.  10.  11.  ++  vii LIST OF ABBREVIATIONS  B/A  basal/apical side  l  microliter  ml  mi 1 1 i 1 i t e r  nM  nanomoles  mM  millimoles  P.E-.  polyethylene  mOsm  milliosmoles  mV  millivolts  hr  hour  mg  milligram  D.W.  dry weight  S.E.  standard e r r o r  P.D.  potential difference  u  K  t  V max  tubing  substrate concentration f o r half-maximal u n i d i r e c t i o n a l f l u x maximal rate of u n i d i r e c t i o n a l f l u x  ACKNOWLEDGEMENTS  I wish to thank my supervisor, Dr. J.E. P h i l l i p s , f o r his guidance and helpful discussions during the preparation o f this thesis.  I am a l s o g r a t e f u l to Ms. Joan Meredith and Dr.  M. Balshin f o r t h e i r assistance.  1  INTRODUCTION  The r o l e of i o n i c r e g u l a t i o n , osmotic r e g u l a t i o n , and  metabolic  waste removal f a l l s mainly upon the Malpighian t u b u l e - r e c t a l complex of most t e r r e s t r i a l  insects [reviewed by Maddrell, 1971].  The process  of  Malpighian tubule secretion and r e c t a l r e s o r p t i o n i s analogous to the glomerular f i l t r a t i o n and tubular r e s o r p t i o n of the vertebrate kidney [Phillips,  1965]. The Malpighian tubules produce a primary f l u i d that i s nearly  iso-osmotic w i t h , but not n e c e s s a r i l y having the same concentrations or proportions of ions and organic molecules as that of the hemolymph [Ramsay, 1956; P h i l l i p s ,  1964c; Maddrell and Klunsuwan, 1973].  The main  d r i v i n g force responsible f o r the production of primary f l u i d i s the +  +  a c t i v e pumping of K  and/or Na  into the tubule lumen.  This transport  of ions and the accompanying flow of water set up electrochemical gradients across the tubule which favor movement of other substances i n t o the tubule lumen [Maddrell, 1971], some amino acids) may  Small organic molecules (sugars,  be brought i n t o the lumen by s o l v e n t - s o l u t e or  s o l u t e - s o l u t e drag, while large organic molecules (organic a c i d s , nitrogenous wastes) may  be a c t i v e l y secreted [Maddrell, 1971].  The rate of f l u i d s e c r e t i o n by the Malpighian tubules of most insects seems to be d i r e c t l y r e l a t e d to the K  +  concentration of  the basal bathing medium [Ramsay, 1956; Berridge, 1968; Maddrell +  Klunsuwan, 1973].  K  and  +  and Na  produce the highest rate of s e c r e t i o n ,  2 but anions also influence s e c r e t i o n . The rate of s e c r e t i o n i s i n v e r s e l y r e l a t e d to the hydrated radius of the secreted anion [Berridge, 1969]. Phosphate i s an exception since i t can support a high rate of s e c r e t i o n completely  disproportionate to i t s hydrated radius [Berridge, 1969]. The primary f l u i d , containing both metabolic wastes and  p h y s i o l o g i c a l l y important s o l u t e s , flows out of the Malpighian  tubules  into the midgut-hindgut j u n c t i o n where i t then flows to the rectum v i a the a n t e r i o r part of the hindgut.  The concentration of the con-  s t i t u e n t s of the primary f l u i d are a l t e r e d to a minor degree by the d i s t a l portions of the Malpighian  tubules in some i n s e c t s and by the  a n t e r i o r portion of the hindgut of others'.  The bulk of the r e s o r p t i o n ,  however, occurs i n the rectum of most insects [Ramsay, 1971]. P h i l l i p s [1964c] found the rectum capable of resorbing over 95% of the primary f l u i d . a c t i v e l y resorbed  Many of the hemolymph constituents are  [monovalent ions -- P h i l l i p s , 1964b, c; amino acids --  Balshin and P h i l l i p s , 1971; water —  P h i l l i p s , 1964a].  The  rectal  epithelium i s covered on the a p i c a l side by a c h i t i n o u s c u t i c l e .  This  c u t i c l e acts as a molecular sieve that prevents the uptake of large molecules including metabolic wastes [ P h i l l i p s and D o c k r i l l , 1968]. Recycling of f l u i d thus r e s u l t s i n the accumulation of metabolic  wastes  i n the rectum due to the a c t i o n of the intima. Since the Malpighian  t u b u l e - r e c t a l complex a l s o c o n t r o l s  osmotic pressure and i o n i c concentrations of Malpighian  of the hemolymph, the rate  tubule secretion i s c l o s e l y c o r r e l a t e d with r e c t a l  reabsorption rates [ P h i l l i p s , 1964c].  Phillips  [1964 a, b, c] studied  Na , K , CI , and water s e c r e t i o n and subsequent r e s o r p t i o n i n the  3  locust and found that i f the balance of ions i n the hemolymph was perturbed, the r e l a t i v e s e c r e t i o n rates of ions by the Malpighian tubules w i l l be out of phase with t h e i r r e l a t i v e rates of reabsorption in the rectum; thus ions i n excess w i l l accumulate i n the rectum and ions i n short supply w i l l be completely reabsorbed. be reabsorbed r a p i d l y under a l l experimental  Water was found to  situations.  Na , K , CI , and water have been the focus of a t t e n t i o n i n ++  most of the i n v e s t i g a t i o n s to date.  PO^, Mg  ++  , and Ca  have been  less i n t e n s e l y s t u d i e d , and then only from the point of view of s e c r e t i o n by the Malpighian tubules. Since PO^ can a f f e c t the rate of production of primary Malpighian f l u i d [Maddrell, and Klunsuwan, 1973] and i s a l s o a metabolic regulator [Levinson, 1971], i t would seem that t h e l l o c u s t should be able to regulate the s e c r e t i o n and reabsorption of PO^ to some extent. Speight [1967] and Maddrell and Klunsuwan [1973] found that PO^ was more concentrated i n the primary Malpighian f l u i d than i n the hemolymph or in the external bathing medium.  Maddrell and Klunsuwan [1973] found  that i n v i t r o preparations of l o c u s t Malpighian tubules were capable of producing a lumen to hemolymph PO^ r a t i o of 1.71 [1969] found that 1.0 mM/1  to 1.0.  Berridge  of arsenate, when added to incubation media  containing PO^ as the most abundant anion, g r e a t l y i n h i b i t e d primary f l u i d production by CalHphora  Malpighian tubules.  Arsenate, aside  from being a metabolic poison, i s a l s o a competitive i n h i b i t o r f o r PO^ c a r r i e r systems [Rothstein, 1963; Levinson, 1971].  This led  Maddrell and Klunsuwan [1973] and Berridge [1969] to suppose that P0  A  4 t r a n s l o c a t i o n may  be f a c i l i t a t e d by some type of c a r r i e r process.  The d i v a l e n t cations Mg  and Ca  secreted by the Malpighian tubules.  do not seem to be a c t i v e l y  Ramsay [1956], while working with  in v i t r o preparations of the Malpighian tubules of the s t i c k i n s e c t , found Mg  and Ca  to be excreted at 1/10  of the two ions i n the bathing medium. above 10 mM/1  to 1/5 of the concentration ++ ++  Concentrations of Mg  i n the bathing media i n h i b i t e d the r a t e of f l u i d s e c r e t i o n  by the Malpighian tubules [Berridge, 1968]. contains C a  and Ca  + +  and M g  The hemolymph, however,  ions a t concentrations more than f o r t y times  ++  greater than i n mammalian blood [Clark and C r a i g , 1953]. authors concluded  that much of the C a  + +  and M g  ++  i s not p h y s i o l o g i c a l l y  a c t i v e i n the hemolymph and i s probably sequestered other organic compounds.  The same  by proteins and  Calcium carbonate i s found i n the Malpighian  tubules of many insects [ C l a r k , 1958], and i s u s u a l l y eliminated as a suspension of granules. l o g i c a l l y a c t i v e Mg  The high concentrations of p o t e n t i a l l y physio-  and Ca  i n the hemolymph may  not necessitate the  reclamation of these ions by the rectum, hence e x c r e t i o n as p r e c i p i t a t e s . The present study was i n mind. 1.  undertaken with two main o b j e c t i v e s  These were: To determine i f Ca  , Mg  , and PO^ are reabsorbed i n the  l o c u s t rectum, as are monovalent ions,, and to compare rates of resorption with rates of s e c r e t i o n of these ions by the Malpighian tubules.  An i n v i t r o preparation, which  i s known to transport monovalent ions and water at rates comparable to those i n v i v o , was  used because of i t s  greater p o t e n t i a l f o r the study of rates under various controlled conditions.  2.  To f i n d the mechanism ( d i f f u s i o n , f a c i l i t a t e d d i f f u s i o n , a c t i v e transport) o f net absorption of any o f these ions. A f t e r doing a b r i e f study on the uptake,of the three i o n s ,  I concentrated on the study of PO^ since i t seemed the most promising as f a r as accumulation was concerned.  The bulk of the t h e s i s i s con-  cerned with the mechanism and l o c a l i z a t i o n o f the uptake system f o r P0„ i n the locust rectum i n v i t r o .  6  MATERIALS AND METHODS  Animals Adult male and female locusts {Schistooeroa  gregaria  Forskal),  from a colony maintained a t 28°C and 60% r e l a t i v e humidity, and f e d a d i e t of bran and l e t t u c e , were used i n a l l experiments. used from one to f i v e weeks a f t e r t h e i r f i n a l molt.  Locusts were  Locusts were starved  f o r 36 hours before an experiment to permit excretion of feces, thus f a c i l i t a t i n g cannulation of the rectum.  Preparation of the Everted Rectal Sacs Cannulated, everted r e c t a l sacs were prepared according to the method of Goh [1971].  The l o c u s t , a f t e r being secured on i t s side  upon a p l a s t i c i n e block beneath a d i s s e c t i n g microscope, was a n a e s t h e t i z ed with a mixture of CO2 and ether.  A U-shaped, d o r s a l - l a t e r a l  was made from the f i f t h to the seventh abdominal segment.  incision  The r e s u l t i n g  f l a p of c u t i c l e was pinned back revealing the rectum with associated tracheae and f a t bodies. away from the rectum.  These structures were c a r e f u l l y dissected  The s l i g h t l y f l a r e d end of a cannula (4 cm of  P.E. 90 tubing) was inserted through the anus and pushed forward u n t i l the f l a r e d end was j u s t a n t e r i o r to the r e c t a l pads.  A l i g a t u r e of  clean human h a i r was used to secure the rectum to the f l a r e d end of the cannula.  The hindgut a n t e r i o r to the cannula was severed from the  7  rectum, and the cannula was slowly withdrawn through the anus u n t i l the ends of the r e c t a l pads emerged.  The everted rectum was then  severed from the body of the l o c u s t . The external surface of the cannulated rectum was r i n s e d i n 10 ml of Ringer s o l u t i o n (Table 1 ) , and the i n t e r n a l surface was flushed with about 0.25 ml of Ringer s o l u t i o n i n j e c t e d through P.E. 10 tubing attached to a syringe.  This procedure removed faecal material or  hemolymph adhering to the rectum.  Another h a i r was used to l i g a t u r e  the open end of the rectum to form a sac attached to a cannula.  A  syringe attached to P.E. 10 tubing was used to remove any f l u i d i n the sac.  The head of a bent i n s e c t p i n was inserted i n t o the end of the  cannula, thus forming a hook by which the r e c t a l sac could be suspended during incubation and weighing, and preventing evaporation of water. The sac was blotted dry with f i l t e r paper and weighed. Twenty ul of Ringer s o l u t i o n were i n j e c t e d into the sac with a 'Hamilton S y r i n g e and the sac was reweighed to obtain the weight 1  at zero-time of incubation. Occasionally a few recta were checked f o r leakage by l e a v i n g them overnight i n an amaranth s o l u t i o n .  Since amaranth cannot penetrate  the intima [ P h i l l i p s and D o c k r i l l , 1968] i t s appearance i n a sac would i n d i c a t e damage to the rectum.  No leakage was observed.  Incubation Procedure The r e c t a l sacs were incubated i n a water bath at a constant temperature (30 ± 0.5°C), and were aerated with a mixture of 95% 0  ?  and  8 5% C0  2  (Figure 1).  The recta were incubated i n 3 - 25 ml of a p i c a l  (external) bathing medium.  Twenty ul of media were used to bath the  basal ( i n t e r n a l ) surface of the r e c t a l p r e p a r a t i o n . For experiments of short duration, sacs were pre-incubated f o r one hour to l e t them achieve a steady s t a t e [Goh, 1971; B a l s h i n , 1973].  At the end of one  hour the contents of the sacs were removed and 20 ul of f r e s h basal bathing media were added.  The sac was then reweighed and t r a n s f e r r e d  t o ' f r e s h apical bathing medium. To determine i f any water was taken up by s w e l l i n g of the r e c t a l t i s s u e , weights of the empty sacs a t zero and f i n a l time ( t ) were compared.  I f any changes occurred, the d i f f e r e n c e was e i t h e r added or  subtracted from the amount of water taken up i n the basal compartment. In t h i s way i t was possible to determine i f net water t r a n s f e r across the r e c t a l sac occurred during the experiment. In a l l experiments, the weighing procedure consisted of removing the sac from the bathing media, b l o t t i n g i t dry (with Whatman No. 1 f i l t e r paper), then weighing i t on a 200 mg t o r s i o n balance.  'August-Sauter'  "  Composition of Bathing Media A Na -Ringer +  (Table 1) was used as the basal bathing medium  and a K -Ringer (Table 1) was used as the a p i c a l bathing medium i n +  most experiments.  The concentration of the ion under study was a l t e r e d  i n some experiments;" such changes are noted i n describing the r e s u l t s . The two Ringers were used i n order to roughly approximate i n vivo  9  Figure 1  Diagram of experimental set-up to study r e c t a l absorption in v i t r o . The everted r e c t a l sac, containing 20 ul of experimental media i n the basal compartment was incubated i n 3-5 ml of apical medium. Mixing and a e r a t i o n was achieved by bubbling 95% 0 and 5% C0 through a p i c a l medium. o  ?  9a  9 5 % O2 5%C0  2  Insect pin Cannula Incubation Vessel Everted Rectal Sac B a s a l Bathing Medium Apical Bathing Medium  10  TABLE 1 Composition of Bathing Media  Constituent  Concentration (gm/1) Na -Ringer +  K -Ringer  Nad  9.82  0.376  KC1  0.48  12.52  MgCl  2  • 6H 0  0.73  0.73  CaCl  2  • 2H 0  0.315  0.315  Dextrose  3.0  3.0  NaHC0  0.18  0.18  0.84  0.84  2  2  3  NaH P0 2  4  Sucrose*  • 1H 0 2  126.17.  168.80  Only added to external media when net water movement was to be prevented.  11 conditions [ P h i l l i p s , 1964b].  For the same reason, the pH of the  external media was set at 5.5 and the i n t e r n a l media was s e t a t pH [Speight, 1967; P h i l l i p s , 1964a].  7.0  A 'Radiometer Model 25' was used to  make a l l pH readings. The pH of the a p i c a l bathing (external) medium during the course of experiments d i d not vary from the zero-time reading.  Measurement of Ion Concentrations i n Media and Tissue Ten ul a l i q u o t s were removed from the a p i c a l and basal bathing media and were analyzed f o r the desired i o n . Inorganic P0^ was measured by the method of Gomori [1942] and the method of Ernster et al_. [1950].  The l a t t e r technique was used f o r  t i s s u e a n a l y s i s , because i t d i d not dephosphorylate l a b i l e organic phosphates as r e a d i l y as d i d the method of Gomori [Martin and Doty, 1949].  A 'Spectronic 20' was used f o r the c o l o r i m e t r i c determinations. Samples f o r M g  ++  determination were put into 3 ml of 0.75%  Na -EDTA and concentrations were determined with a 'Techtron 120AA' +  flame  spectrophotometer [as per Unicam Method Sheet, 1967]. Calcium-45 samples were counted on a 'Nuclear Chicago Mark I  l i q u i d s c i n t i l l a t i o n counter.  1  1-3 ul samples were c o l l e c t e d with  'Drummond Microcaps' micropipettes.  These samples were put into 10 ml  •of e i t h e r Bray's s o l u t i o n [Bray, 1960] or Aquasol (New England Nuclear) f o r counting. 32 F l u i d samples (1-3 u l ) containing  P were placed on planchets,  dried and then counted on a 'Nuclear Chicago Model 1042' automatic planchet counter.  12 32 Tissues t o be analyzed f o r P were f i r s t r i n s e d with i s o osmotic mannitol and were b l o t t e d dry with f i l t e r paper. The r e c t a l t i s s u e was then spread out evenly on a planchet and was d r i e d slowly under an i n f r a - r e d lamp.  The planchet w i t h the d r i e d t i s s u e was counted  on a 'Nuclear Chicago Model 1042' planchet counter. 45 Tissues analyzed f o r  Ca content were rinsed with i s o -  osmotic mannitol, b l o t t e d , put i n t o weighed platinum boats, and were weighed.  The tissues were d r i e d f o r 48 hours a t 80°C i n a drying oven.  The dry weights were found, and then the t i s s u e s were dry ashed i n a muffle furnace a t 460°C f o r 24 hours [ P h i l l i p s , 1964b].  The ash was  dissolved i n 1 ml of d i s t i l l e d water and 3-25 ul samples were taken with Lang-Levy p i p e t t e s .  These samples were put into 10 ml o f s c i n t i l l a t i o n  f l u i d and counted as described above. Tissues to be analyzed f o r Mg were ashed by the same pro++ + cedure as f o r Ca . The ash was d i s s o l v e d i n 3 ml of 0.75% Na -EDTA and the M g content was read on the flame spectrophotometer ('Techtron ++  120AA') i n the atomic absorption mode of operation. Inorganic phosphate with i c e cold 10% TCA.  ( P i ) was extracted from a t i s s u e homogenate  The p r e c i p i t a t e was c e n t r i f u g e d , washed with  more i c e cold 10% TCA, then centrifuged again.  The supernatant was  decanted and analyzed f o r P i by the method of Ernster e t a]_. [1950].  E l e c t r o p o t e n t i a l Differences Across the Rectal Sac In order to determine the d i r e c t i o n and the magnitude of the p o t e n t i a l d i f f e r e n c e (P.D.) across the r e c t a l sac, the apparatus shown i n Figure 2  was used . A 'Keithly Model 602' electrometer was used  13  Figure 2  Apparatus used f o r measurement o f t r a n s r e c t a l  potentials.  The asymmetry potential was obtained by immersing the the basal end of the KC1 bridge into the a p i c a l medium.  KEITHLY  MODEL 602 ELECTROMETER  Calomel Electrode 9 5 % 02 5 % C 0  3M/L KCl Bridge  2  Basal End of KCl Bridge Everted Rectal Sac Apical Bathing Medium 3M/L KCl —  14 to measure potential d i f f e r e n c e s .  'Radiometer' calomel electrodes i n  s e r i e s with a 3 M/l KCl-agar bridge made up i n P.E. 10 tubing completed the c i r c u i t .  The asymmetry p o t e n t i a l d i f f e r e n c e was found by i n s e r t i n g  the agar bridge, shown on the basal s i d e , i n t o the vessel the a p i c a l bathing medium.  containing  The asymmetry p o t e n t i a l was subtracted from  the measured t r a n s - r e c t a l p o t e n t i a l  differences.  Treatment of Results The Student's t - t e s t was used to s t a t i s t i c a l l y t e s t f o r s i g n i f i c a n c e of the observed d i f f e r e n c e s .  Unless the p r o b a b i l i t y l e v e l  i s s p e c i f i c a l l y stated, the term " s i g n i f i c a n t l y d i f f e r e n t " has a p r o b a b i l i t y less than or equal to 0.05.  15  Figure 3  Trans-rectal p o t e n t i a l d i f f e r e n c e s ( a p i c a l s i d e p o s i t i v e ) across everted r e c t a l sacs with time a f t e r preparation. Na-Ringer was present i n both compartments (apical and basal) and 420 mOsm/1 of sucrose was added to the a p i c a l medium to prevent water movement. The v e r t i c a l bars represent ±S.E. of the mean (7 preparations).  POTENTIAL DIFFERENCE (mV) 00 CD ^4 o o O O o  4S  o o  m  \  to  X o  C/)  0  \ \  00 O T  CO  o  16  RESULTS  V i a b i l i t y of the i n i v i t r o Rectal Preparation Goh [1971] evaluated the type o f i n v i t r o preparation used i n t h i s study.  His r e s u l t s were comparable to those observed i n vivo +  +  by P h i l l i p s [1964b] with respect to the a c t i v e t r a n s p o r t of Na , K , C l ~ , and H^O.  Balshin [1973] used the same type of everted i n v i t r o  preparation to demonstrate a c t i v e transport of amino a c i d s .  He used the  e l e c t r o p o t e n t i a l d i f f e r e n c e across the r e c t a l w a l l , and the uptake r a t e of H 0 to assess the s t a b i l i t y of the preparation with time. 2  A  steady s t a t e c o n d i t i o n was a t t a i n e d a f t e r one hour o f preincubation and was maintained  f o r a t l e a s t 6 hours.  The preparations used i n t h i s  study e x h i b i t e d the same degree o f s t a b i l i t y with regard to P.D. (Figure 3) and H 0 uptake rate (Figure 4) f o r a t l e a s t 6 hours, the longest period 2  of time during which experiments were conducted. Water movement into the r e c t a l sac was blocked or decreased i n some experiments by the a d d i t i o n o f sucrose (a non-permeating molecule [ P h i l l i p s and D o c k r i l l , 1968] to the a p i c a l bathing medium.  Na -Ringer +  requires 420 mOsm/l of sucrose i n the a p i c a l bathing medium to block H 0 movement [ B a l s h i n , 1973], but K -Ringer requires 586 mOsm/l of +  2  sucrose to block H 0 movement (Figure 4). 2  This seems to i n d i c a t e that  the preparation can remove H 0 more e f f e c t i v e l y from a K -Ringer than +  2  from a Ma -Ringer.  Water i s also absorbed a t a s l i g h t l y higher rate  17 from K -Ringer (9.8 ul/hr/rectum, Figure 4) as compared to Na -Ringer +  +  (7.2 ul/hr/rectum, B a l s h i n , 1973] i n the absence of an osmotic d i f f e r e n c e across the r e c t a l sac. The i n i t i a l  p o t e n t i a l d i f f e r e n c e f e l l s l i g h t l y a f t e r the f i r s t  hour of incubation and then remained between 50 and 60 mV, lumen p o s i t i v e , f o r the next 5 hours.  The observed P.D. was of the same  p o l a r i t y , but greater than that observed i n vivo [ P h i l l i p s , 1964b]. Goh [1971] showed that i n vivo rectum was capable of producing a P.D. o f the magnitude observed i n v i t r o under c e r t a i n c o n d i t i o n s .  The s t a b i l i t y  and magnitude of the observed P.D. (50-60 mV, lumen p o s i t i v e ) agrees c l o s e l y with that measured i n v i t r o by Balshin [1973] during the 5 hour steady-state period.  ++  Test f o r Accumulation of Ca  , Mg  ++  , and P0^  To see i f these inorganic ions accumulated across the r e c t a l w a l l , equal concentrations of the ion were placed on both sides of the everted r e c t a l sacs a t zero-time.  Accumulation of ions with time  would i n d i c a t e some d r i v i n g force other than concentration d i f f e r e n c e . Factors such as solvent drag and P.D. could then be examined to see i f they could account f o r the accumulation.  I f solvent flow i s prevented  and the P.D. can not account f o r an observed accumulation, a c t i v e transport might then be postulated. ++  The concentrations o f f r e e (non-precipitated) Mg  ++  and Ca  in the urine of adult locusts have not been measured, but are expected to be low [1mm Ca  /!, 9mm Mg  / I f o r the s t i c k i n s e c t ; Ramsey, 1956].  18  Figure 4  The net rate of water movement across the r e c t a l wall i n the absence and presence of an osmotic gradient. The baching medium i n the a p i c a l compartment was K -Ringer, and Na -Ringer was present as the basal medium. ( 0 ) 586 mOsm/l of sucrose was present i n the apical medium; ( H ) no sucrose was present i n the a p i c a l medium. V e r t i c a l bars represent ±S.E. of the mean (4 preparations).  19 The Ringer concentrations (Ca  2.14 mM/1, Mg  3.59 mM/1) o f these  ions were therefore considered appropriate to t e s t f o r net t r a n s f e r .  Net C a  + +  Movement Basal to a p i c a l concentration r a t i o s (B/A) o f C a  + +  did not  vary from u n i t y a f t e r 3 hours o f incubation i n the absence o f water movement (Figure 5).  Experiments were conducted a t two other media ++  concentrations (0.2 and 0.02 mM/1 of Ca  ) but again B/A r a t i o s did not  exceed u n i t y , i n d i c a t i n g only a minimum o f Ca movement (Figure 5). When C a was placed i n i t i a l l y only i n the a p i c a l medium ( u n i d i r e c t i o n a l 4 5  f l u x experiment), a small amount (0.92 ± 0 . 2 nm/mg D.W. r e c t a l t i s s u e ) of C a  was observed i n the basal medium a f t e r 3 hours o f i n c u b a t i o n ,  + +  i n d i c a t i n g that the r e c t a l wall i s only s l i g h t l y permeable to Ca from the a p i c a l side (Table 2). When the recta are incubated i n 0.2 mM/1 ++ 45 of Ca , with t r a c e r on both s i d e s , 4 times more Ca enters the r e c t a l t i s s u e than under s i m i l a r conditions when t r a c e r i s placed only on the apical side.  This i n d i c a t e s that C a  enters the rectum from the basal  + +  side much more r a p i d l y than from the a p i c a l side (Table 2).  Net M g  ++  Accumulation In these experiments the M g  ++  concentration i n the basal  bathing medium was i n i t i a l l y 3.6 mM/1 while the M g  ++  concentration i n  the a p i c a l bathing medium was varied over a 100-fold range.  The B/A  concentration r a t i o s were c a l c u l a t e d from the average hourly rates of  20  Figure 5  The net movement of Ca  and water across the r e c t a l w a l l ,  a f t e r 3 hours of incubation i n Ringer containing various ++ amounts of Ca K -Ringer was present as the a p i c a l bathing medium (with 586 mOsm/1 of sucrose), and Na -Ringer was present as the basal bathing medium. At zero-time the C a concent r a t i o n s were the same i n both the a p i c a l and basal bathing media (0.02, 0.2, or 2.0 mM/1 o f C a ) . Preparations were pre-incubated f o r one hour i n the experiment a l media. The v e r t i c a l bars represent ±S.E. of the mean (5 preparations). + +  + +  20a  L U CO  °<  L L . H O O L U U L ! Qn  <§? t ° LU  c  •7 C  0 "1  *2r SJJ  < UJQ2 3  0 -2 INITIAL  002 0-2 [Ca ] I N A P I C A L MEDIUM(mM/L) ++  20 BATHING  21 TABLE 2 The Net Movement of C a i n t o the Rectal Tissue and Into the Basal Bathing Medium, Over 3 Hours of Incubatioiji in Media Containing Various Amounts of Ca + +  +  K -Ringer with various C a concentrations was the a p i c a l bathing medium and Na -Ringer, with i n i t i a l C a concentration i d e n t i c a l to t h a t of the apical bathing medium, was the basal bathing medium. 586 mOsm/l of sucrose was added to the a p i c a l bathing medium to prevent water movement. Recta were pre-incubated f o r one hour i n the experimental media. The r e s u l t s are expressed as the mean ±S.E. (at l e a s t 5 p r e p a r a t i o n s ) . + +  + +  I n i t i a l Apical and Basal Ca Concentration + +  ("W )  Net Accumulation of C a (nM/mg D.W. Rectal Tissue) . + +  In Basal Compartment  1  In Rectal Tissue  0.02  0.04 ± 0.02  0.5 ± 0.1  0.2  1.08 ± 0.6  6.6 ±1.1  2.0  *  0.2 (unidirectional  •1.5  ± 1.0  17.9 ± 3.5  0.9  ± 0.2  1.4 ± 0.1  flux)  u n i d i r e c t i o n a l f l u x -- 0.2 mM/1 of C a was present i n the a p i c a l and basal bathing medium, but t r a c e r ( C a ) was present only i n the a p i c a l bathing medium. + +  4o  22 net uptake (Figure 6).  Since these rates were so s m a l l , the B/A  r a t i o barely exceeded u n i t y , i n d i c a t i n g v i r t u a l l y no basal M g accumulation. Figure 6. ++  ++  Two other observations of i n t e r e s t are evident from F i r s t l y , the i n f l u x o f water has no s i g n i f i c a n t e f f e c t on ++  the Mg  uptake rate except a t 36 mM/1 o f Mg  i n the a p i c a l  bathing  medium.  Secondly, a t e n f o l d a p i c a l to basal concentration d i f f e r e n c e +4*  has no s i g n i f i c a n t e f f e c t on the uptake of Mg  i n the basal compartment  when water movement i s prevented. Figure 7 i n d i c a t e s that the r e c t a l t i s s u e content o f M g ++ i s r e l a t i v e l y independent o f Mg medium.  concentration i n the a p i c a l  ++  bathing  These measurements were made a t the end o f four hours o f  incubation.  Freshly e x t i r p a t e d , recta contain 4.8 ± 1.5 nm Mg /mg ++  D.W. r e c t a l t i s s u e . Net PO4 Accumulation Speight [1967] measured the PO^ concentration o f hindgut f l u i d (made up mostly o f Malpighian tubule f l u i d ) , and deproteinized hemolymph o f l o c u s t s .  She found conc^ja^fcrtions o f 14.6 ± 5.6 and  6.2 ± 1.3 mM/1 of PO4 (mean ±S.D.) r e s p e c t i v e l y . Rectal concentration of  P0  4  was 42 mM/1. With these i n vivo PO^ concentrations i n mind, r e c t a l pre-  parations were incubated i n Ringers containing e i t h e r 4, 12 or 42 mM/1 of PO^ i n both a p i c a l and basal bathing media.  In Figures 8 and  9 the change i n PO^ concentration r a t i o s ( B a s a l / a p i c a l s i d e ; B/A) i s p l o t t e d against time, and against PCL concentration i n the a p i c a l  23  Figure 6  The net movement of M g various M g  ++  ++  and H^O across the r e c t a l wall a t  concentrations i n the a p i c a l bathing medium,  with and without net water movement across the r e c t a l w a l l . K -Ringer with various Mg concentrations was present as the a p i c a l bathing medium, with 586m0sm/l of sucrose ( 0 ), or without sucrose ( H )• Na -Ringer was present as the basal bathing medium. The i n i t i a l M g concentration i n the basal compartment (3.6 mM/1) was the same i n a l l e x p e r i ments. The rates are an average of hourly rate measurements taken over 3 hours. Preparations were preincubated f o r one hour i n the experimental media. The v e r t i c a l bars represent ±S.E. of the mean (4 preparations). +  ++  23a  LJJ  CO CO  260  <  Ui  &50 ^40 Q30  O ov lli E20 10 < or E 0 c  uj^  -10  1 5  ! | 10 U- LU  c 0  Oh o  v :  3  0 - 1 0  20  30  40  [Mg* -5 ] IN APICAL BATHING MEDIUM(mM/l) 4  24  Figure 7  The t o t a l amount of Mg  +  i n r e c t a l t i s s u e a f t e r 4 hours  of incubation, v/ith and without sucrose present i n the apical bathing medium. K -Ringer with various Mg concentrations was the a p i c a l bathing medium, with 586 mOsm/1 of sucrose ( 0 ) or without sucrose added ( @ ) . Na -Ringer was the basal bathing medium, with an i n i t i a l concentration of 3.6 mM/1 of M g i n a l l experiments. The v e r t i c a l bars represent ±S.E. of the mean (4 preparations). +  ++  Mg * +  C O N T E N " OF RECTA  (nm/mg D.W. R O  Y  CTAL  o  TISSUE)  4N O  —I  25 bathing medium, r e s p e c t i v e l y . i n 2 hours and was maintained  The highest B/A r a t i o (ca. 3) was obtained f o r a t l e a s t 4 more hours i n an experiment  where 4 mM/1 PO^ was placed i n both a p i c a l and basal bathing media a t zero-time.  Preparations incubated i n both 12 and 42 mM/1 o f PO^ showed  a gradual increase i n B/A with time, but neither concentration could produce a B/A r a t i o as great as that observed when 4 mM/1 of PO^ was present.  Tissue Contribution to Uptake of PO^ i n the Basal  Compartment  In order to determine how much, i f any, PO^ was contributed to the basal compartment by the t i s s u e , preparations were incubated f o r 6 hours i n PO^-free medium with 586 mOsm/l of sucrose i n the a p i c a l bathing medium.  During t h i s period 114.8 ± 14.7 nm PO^ rectum (mean  ±S.E.) accumulated i n the basal compartment. the t o t a l accumulation  This was almost as much as  of PO^ (139.0 ± 25.3 nm/retum) a t 6 hours i n the  basal compartment when the recta were incubated i n a p i c a l and basal bathing media containing i n i t i a l l y 7 mM/1 of P0^.  In another experiment  when 6.0 mM/1 of P0^ was present i n the basal bathing medium and 0.6 mM/1 of PO^ was present i n the a p i c a l bathing medium, a t o t a l of 60 nM/rectum of P0^ accumulated i n the basal compartment a f t e r 6 hours of incubation.  * I t seems, that when the basal bathing medium contains  l i t t l e or no P0^, the r e c t a l t i s s u e can contribute s u b s t a n t i a l amounts of P0^ to the basal compartment.  Since the P0^ accumulation  i n the basal compartment increases with increasing P0^ concentration i n the apical bathing medium, when the PO^ concentration i n the basal  26  Figure 8  The basal:  a p i c a l (B/A) concentration r a t i o of P 0  4  across  i n v i t r o r e c t a l sacs with time. K -Ringer was the a p i c a l bathing medium, and Na -Ringer was the basal bathing medium. The a p i c a l and basal bathing media both contained i n i t i a l l y e i t h e r 4( © ), 12 ( © ), or 42. ( A ) mM/1 of P0 . The a p i c a l bathing medium contained 586 mOsm/1 of sucrose to prevent water movement. The v e r t i c a l bars represent ±S.E. o f the mean (4 preparations). 4  -0  26a  27  Figure 9 The f i n a l PO4 r a t i o (B/A) and the net movement o f water across the r e c t a l sacs ( a f t e r 6 hours of incubation) when the PO^ concentration of the bathing media was varied. + + K -Ringer was the a p i c a l bathing medium and Na -Ringer was the basal bathing medium. The a p i c a l and basal bathing media both contained i n i t i a l l y e i t h e r 4, 12, o r 42 mM/1 of PO4. The a p i c a l bathing medium contained 586 mOsm/l of sucrose. The v e r t i c a l bars represent ±S.E. o f the mean (4 preparations).  1  D_  0 cr+4 L  U  "T,  ^v: <  0  LUCL  -4  0 10, 2 0 30 4 0 45 INITIAL[P0 ] IN APICAL AND BASAL BATHING MEDIA (mM/D r  4  28 compartment i s i n i t i a l l y held constant, at l e a s t part of the accumul a t i o n i s due to t r a n s e p i t h e l i a l movement of PO^ against a large gradient.  Type of PO^  (Inorganic and/or Organic) i n the Basal Compartment  I t was of i n t e r e s t to see i f the PO^ accumulating basal compartment was a l l inorganic.  i n the  The method of Ernster et aj_. [1950]  was used to measure the Pi present i n an a l i q u o t drawn from the basal bathing medium.  Another a l i q u o t from the same medium was  hydrolyzed  hot a c i d , thus cleaving PO^ from a c i d - l a b i l e organic phosphates. was then measured by the method of Ernster et al_. [1950].  with  The  PO^  The amount  of PO^ did not increase over the amount of inorganic phosphate measured in the f i r s t a l i q u o t ; hence, a l l the measurable PO^ i n the basal  bathing  medium was present as inorganic phosphate. 32 A s i m i l a r t e s t was conducted when  P was used to estimate  the rate of accumulation of PO^ i n the basal compartment [Ernster et a l . 1950].  The method of Ernster et al_. [1950] was used to remove a l l the 32  Pi  (including  medium.  P i ) from an a l i q u o t taken from a sample of basal bathing 32 The same a l i q u o t with the Pi removed was then counted on a  clanchet counter.  Only the background reading was observed, i n d i c a t i n g  32 that no  P  was present i n an organic form.  29 E f f e c t of Water Uptake on PO^ Accumulation i n the Basal Medium To t e s t the e f f e c t of r^O uptake on the accumulation  of PO^  i n the basal compartment, recta were incubated i n Ringer with no sucrose present, hence no osmotic d i f f e r e n c e e x i s t e d between the a p i c a l and basal compartments.  No d i f f e r e n c e i n the r a t e of PO^ uptake i n  the basal compartment was observed between recta incubated i n the absence or presence of an osmotic gradient, when the PO^ concentration i n the a p i c a l bathing medium was low (Figure 10). However, a t the highest PO^ concentration i n the a p i c a l medium (61 mM/1), H 0 uptake 2  has a s i g n i f i c a n t (p < 0.001) e f f e c t on the r a t e of PO^ t r a n s f e r i n t o the basal compartment.  Kinetics Saturation k i n e t i c s are obtained when the net rate of PO^ uptake i n the basal compartment i s p l o t t e d against a 100~fold external concentration range (0.6 - 61.0 mM/1) of PO^ (Figure 10). The zerotime concentration of PO^ i n the basal compartment was 6.0 mM/1 of PO^ i n a l l experiments.  The data f i t Michealis-Menton  k i n e t i c s with K  t  of 5.0 mM/1 and V of 52.6 nM/hr/mg D.W. r e c t a l t i s s u e (Figure 11). max Saturation k i n e t i c s i s a phenomenom associated with c a r r i e r mediated processes.  I f PO^ was d i f f u s i n g i n t o the basal compartment,  a l i n e a r r e l a t i o n s h i p (Fick's Law) should occur when rate i s p l o t t e d against the PO^ concentration i n the a p i c a l bathing medium. not the case.  This was  S t e i n [1967] considers s a t u r a t i o n as " r e l a t i v e l y strong"  evidence f o r carrier-mediated systems.  30  Figure 1 0  The net rate of P 0 ^ appearance i n the basal compartment and the net water movement across the r e c t a l sac f o r a range of PO^ concentrations i n the a p i c a l  bathing  medium. K -Ringer with varied P 0 » concentrations was the a p i c a l bathing medium, with 420 mOsm/l o f sucrose present ( © ) and without sucrose present ( @ ). Na -Ringer was the basal bathing medium, that contained i n i t i a l l y 6 . 0 mM/1 of PO, i n a l l experiments. Net rate o f P O 4 appearance i n th§ basal compartment was measured over a period o f 1 hour. The v e r t i c a l bars represent ±S.E. o f the mean (at l e a s t 4 preparations). +  +  30a  ~6Qr  LLJ  3  o)50  ^ £  LLV  o<  2 5 r  10 0-  0  0  [PO4]  0  10 20 30 60 IN APICAL BATHING MEDIA(mM/l)  31  Figure 11  Double r e c i p r o c a l p l o t of the net rate of PO^ entry into the basal compartment as a f u n c t i o n o f the PO^ concentration in the a p i c a l bathing medium. This p l o t was drawn from the values obtained from Figure 10. Water movement was r e s t r i c t e d with 420 mOsm/1 of sucrose i n the a p i c a l bathing medium.  31a  32  I n h i b i t i o n of Phosphate Transfer Another t e s t f o r c a r r i e r mediated systems involves uptake rate measurements i n the presence of substrate analogs.  Analogs compete with  a natural substrate f o r a c a r r i e r s i t e and t h i s competition shows up as a decreased rate of uptake of the natural substrate.  Rothstein [1963]  demonstrated that arsenate was a competitive i n h i b i t o r f o r the a c t i v e PO^ uptake system i n yeast c e l l s .  Berridge [1969], found that arsenate  i n h i b i t e d PO^ driven s e c r e t i o n by the Malpighian tubules of an i n s e c t , and postulated that the arsenate was competing (with PO^) f o r a c a r r i e r s i t e on the membrane. Recta were incubated i n Ringer containing 8 mM/1 8 mM/1  of AsO^ i n the a p i c a l and basal bathing media.  of PO^ and  586 mOsm/l of  sucrose was present i n the a p i c a l bathing medium to prevent h^O movement. 32 The t o t a l amount of  P that entered into the t i s s u e over a 6 hour period  was measured a f t e r incubation i n the absence and i n the presence of AsO^.  Twice as much PO^ accumulated i n the control t i s s u e (p <  i n d i c a t i n g AsO^ i n h i b i t s P0^ uptake (Table 3).  0.01)  However, as well as  being a competitive i n h i b i t o r f o r PO^ uptake systems, AsO^ i s also a metabolic poison; so a t t h i s point i t was not p o s s i b l e to t e l l  whether  the AsO^ was i n h i b i t i n g a PO^ c a r r i e r , or whether the AsO^ was a f f e c t ing c e l l u l a r energy metabolism and hence PO^ uptake. A combination of 2 mM/1 iodoacetic acid with 5 mM/1  of potassium cyanide and 2 mM/1  of  of PIPES buffer (piperazine-N, N-bis (2-  ethane s u l f o n i c acid)" monosodium monohydrate) and 50 mOsm/l of sucrose i n the a p i c a l bathing medium at pH 6.6 was used to a b o l i s h water uptake in the recta [Balshin and P h i l l i p s , 1971],  The  control  media  33 TABLE 3 Net  32 P Uptake i n Either Control o r Poisoned Tissue, A f t e r 6 Hours o f Incubation  In experiment 1, K -Ringer with 586 mOsm/1 o f sucrose, 8 mM/1 o f P0», and 8 mM/1 of AsCL was the experimental bathing medium (pH 5.5), and Na -Ringer with 8 mM/1 or PG\ was the basal bathing medium (pH 7.0). The bathing media were the same f o r the control except f o r the absence of A S O 4 . In experiment 2, Na -Ringer with 5 mM/1 o f PIPES buffer, and 8 mM/1 of P0» was present as the a p i c a l and basal bathing medium ( c o n t r o l ) . The a p i c a l bathing medium contained 420 mOsm/1 of sucrose. The experimental bathing media.were the same as the control bathing media with the a d d i t i o n o f 2 mM/1 o f KCN and 2 mM/1 o f IAA to both bathing media. The a p i c a l bathipg medium (pH 6.6) contained 50 mOsm/1 of sucrose. The r e s u l t s are expressed as the mean ±S.E (6 preparations). +  +  +  Experiment  Apical Bathing Medium  PO4/rectum)  Net H?0 Movement ( u l / rectum)  586  107.3 ± 8.6  +1.4 ± 3.4  Sucrose i n Apical Bathing Medium (mOsm/1)  Tissue Uptake of 32p ( M n  1  K -Ringer  1  K -Ringer plus 8mM/l of A s 0  .586  56.9 ± 4.9  -5.4 ± 6.0  2  Na -Ringer  420  '64.6 ±10.2  +2.6 ± 1.2  2  Na -Ringer plus 2mM/l of KCN and IAA  47.4 ± 6.7  -0.2 ± 2.4  +  +  4  +  +  50  34 contained 420 mOsm/l of sucrose i n the a p i c a l bathing medium to a b o l i s h h^O uptake.  A f t e r 6 hours o f incubation (no p r e - i n c u b a t i o n ) , 32  no s t a t i s t i c a l d i f f e r e n c e (p > 0.1) i n t i s s u e  P uptake could be  observed between the control and t e s t preparations.  Seemingly, P0^  entry into the r e c t a l t i s s u e from the a p i c a l bathing medium i s not a f f e c t e d by the presence of KCN and IAA i n the a p i c a l  bathing  medium. Since the  P accumulation  was measured i n the t i s s u e , the  a p i c a l membrane i s the only b a r r i e r between the t i s s u e and the a p i c a l bathing medium.  Therefore, unless AsO^ was i n t e r f e r i n g with i n t r a -  c e l l u l a r PO^ binding, the experiments with AsO^ and the metabolic i n h i b i t o r s KCN and IAA may i n d i c a t e that the l o c a t i o n of the c a r r i e r i s i n the a p i c a l membrane. Further C h a r a c t e r i z a t i o n of the System The amount of P0^ taken up i n t o the basal compartment, when 32 measured chemically, exceeded that estimated by  P uptake by about a  f a c t o r of 4 (Table 4). This observation prompted an i n v e s t i g a t i o n i n t o the t i s s u e incorporation of P0^ into organic forms during incubation i n 3 2  P labelled P0 . 4  The Pi and the a c i d - l a b i l e organic-PO^ content of f r e s h l y extirpated unincubated frt aj_. [1950].  recta were determined using the method o f Ernster  Another group of recta were then incubated i n Na-Ringer  with 5 mM/1 of PIPES buffer a t pH 6.6.  This modified Na-Ringer was 32 the apical and basal bathing medium with the a d d i t i o n of P to the  35 TABLE 4 Various Fractions of PC *, Determined by Chemical Analysis and 1  Radiotracer Estimation ( P ) , i n Incubated and Unincubated Recta Recta were incubated f o r 6 hours. Na -Ringer with 5 mM/1 o f PIPES buffer and 8 mM/1 o f PO. was the incubation medium i n both the a p i c a l and basal compartments f o r control t i s s u e . In a d d i t i o n , the a p i c a l bathing medium (pH 6.6) contained 420 mOsm/1 of sucrose to prevent net water movement. The incubation media f o r the poisoned incubated t i s s u e s were the same as f o r the control t i s s u e s with the a d d i t i o n o f 2 mM/1 of KCN and 2 mM/1 of IAA to both bathing compartments. The incubation medium on the a p i c a l side (pH'6.6) contained 50 mOsm/1 o f sucrose. Results are expressed as the mean ±S.E. (6-12 preparations).  Method of  Treatment of PO4 Recta Determination  Inorganic Acid P0 (Pi) Labile (nM/rectum) OrganicPO4 (nM/ rectum) 4  Acid Labile OrganicPO4 plus Pi (nM/ rectum)  Non-Acid Labile OrganicP0 (nM/ rectum) 4  Net Basal Uptake of P 0 (nM/ rectum) 4  urn ncubated P Estimation 3 2  Chemical Analysis  incubated control  20.5±2.1  45.6  incubated poisoned  44.8±6.9  7.8+1.1  unincubated  52.2±4.5  incubated control  115.2± 6.1  incubated poisoned  173.2± 11.7  66.H6.9  16.2±0.8  24.0±2.8  29.8±3.1  66.0 118.2± 5.8 149.0 264.2± 11.0  94.9±8.1  65.0±4.4  36 a p i c a l bathing medium only.  A f t e r being incubated f o r 6 hour, the  tissues were analyzed f o r Pi and acid l a b i l e organic-PC^.  The chemical  determinations showed a s i g n i f i c a n t 2-fold increase i n both Pi and acid l a b i l e organic-PO^ over the unincubated t i s s u e s . 32 The values derived f o r  P i n f l u x i n d i c a t e that roughly three-  quarters of the PO^ taken up by the r e c t a l t i s s u e (61.8 nM/rectum) goes into organic-PC^, while one-quarter (20.5 nM/rectum)remains as P i . This 32 amount of  P accounts f o r about one-third of the increase i n chemically  determined Pi over the amount obtained from unincubated r e c t a .  The  a d d i t i o n a l amount may occur as a by-product of energy metabolism, and possibly as a release of previously sequestered unlabelled  PO^.  I f preparations are incubated i n the presence of 2 mM/1 and 2 mM/1  of KCN  of IAA, the chemically determined t i s s u e Pi increases more  than 3 f o l d (173.2 ± 11.7 nM/rectum) over the unincubated t i s s u e , and about 1.5 times (115.2 ± 6.08 nM/rectum) over the amount of Pi i n the 32 control t i s s u e . The P estimate more than doubles (44.8 ± 6.9 nM/ 32  rectum) over the control value (20.5 ± 2.1 nM/rectum).  The  P estimate  of a c i d - l a b i l e organic-PO^ (7.78 ± 1.1 nM/rectum) i n the poisoned t i s s u e i s about one-sixth of the amount of organic-PO^ i n the control t i s s u e (45.6 nM/rectum), i n d i c a t i n g that the metabolic i n h i b i t o r s have i n t e r f e r r e d with the metabolic incorporation of P0^ into organic forms, but have not i n t e r f e r r e d with the entry of P0^ into the t i s s u e . The chemically determined amount of P0^ i n the basal compartment was s i g n i f i c a n t l y greater f o r the control recta (94.9 ± 8.1 nM/rectum) 32  than f o r the poisoned recta (65.0 ± 4.4 nM/rectum).  However, the  P  37  estimations of the amount of PO^ accumulated i n the basal compartments of poisoned recta were almost i d e n t i c a l with the estimations of the 32  amount of PO^ accumulated by the control recta (also determined by estimation).  P  This observation seems to i m p l i c a t e metabolism i n the  accumulation of PO^ i n the basal compartment.  Metabolic release of PO^  32  leads to a decrease i n  PO^ s p e c i f i c a c t i v i t y i n the t i s s u e , hence i t  i s not s u r p r i s i n g that chemical t i o n i n the basal compartment do  and  i s o t o p i c estimations of PO^ accumula-  not'agree.  38  DISCUSSION  In this' study an i_n v i t r o preparation was used to determine ++  whether the locust rectum i s p o s s i b l y a s i t e of Mg ++  reabsorption and hence r e g u l a t i o n . Ca  ++  , Ca  , and  PO^  4*4"  and Mg  (at low concentrations)  do" not appear to be transported by the i n v i t r o rectum.  PO^ i s  accumulated i n the basal compartment against a s i z e a b l e electrochemical gradient. These f i n d i n g s are perhaps not s u r p r i s i n g when observations from the l i t e r a t u r e are considered.  Urate, carbonate, o x a l a t e , and  phosphate s a l t s of the d i v a l e n t cations are found as p r e c i p i t a t e s i n the lumen of the Malpighian tubules of many i n s e c t s , i n c l u d i n g Orthopterans [reviewed by C l a r k , 1958].  Ramsay [1956], using an i n v i t r o  preparation of s t i c k i n s e c t (Orthopteran) Malpighian tubules, found ++  ++  that both Mg  and Ca  are present at much lower concentrations i n  Malpighian tubule f l u i d than i n the hemolymph.  In t h i s study, i t was  found that the i n v i t r o l o c u s t rectum has a very low permeability to Ca  + +  from the a p i c a l s i d e , but C a  + +  can enter the r e c t a l t i s s u e from  the basal side much more r e a d i l y (Table 2). suggest that Ca  The l a t t e r observation  may  i s exchanged between the storage s i t e s w i t h i n t i s s u e s  and the hemolymph, thus maintaining a dynamic balance of f r e e C a the hemolymph of the l o c u s t .  The amount of unbound C a  + +  within  + +  i n the hemolymph  i s u l t i m a t e l y regulated by the Malpighian tubules that secrete C a  + +  ++ low r a t e s .  Ca  may  be continuously absorbed ( a c t u a l l y concentrated  ++ as i s Mg  ; Wyatt, 1961) from digested food material i n such large  at  39  q u a n t i t i e s as to insure a steady supply o f t h i s d i v a l e n t c a t i o n . In ++ e f f e c t , excess of Ca may be the normal s i t u a t i o n , and d e f i c i e n c i e s e x c e p t i o n a l l y rare.  -  ++ ++ The apparent lack of Mg uptake (at low Mg concentrations) by i n v i t r o l o c u s t recta may r e f l e c t the i n vivo s i t u a t i o n , since r e g u l a t i o n (as suggested f o r C a ) perhaps occurs a t the t i s s u e and Malpighian ++  tubule l e v e l s .  At the hemolymph l e v e l a dynamic balance between bound (a  f a i r l y large amount; Wyatt, 1961) control the amount o f free M g ++ t o t a l Mg fluctuation. amount of the t o t a l M g  ++  and p h y s i o l o g i c a l l y a c t i v e M g  ++  could  present throughout quite a wide range o f  The Malpighian tubules excrete only a small ++  present i n the hemolymph [Ramsay, 1956].  This  observation coupled with the observation that almost a l l i n s e c t s , concen++ t r a t e Mg  from t h e i r food [Wyatt, 1961] may not necessitate a c t i v e  retention of M g  ++  by the rectum.  PO^ does seem to be recycled because i t i s resorbed by the in v i t r o l o c u s t rectum.  PO^ i s accumulated as P i i n the basal compartment,  and the rate of uptake i s a f f e c t e d by the phosphate concentrations i n the apical and basal bathing media. and chemical  This uptake i s against both e l e c t r i c a l  gradients, cannot be explained by solvent drag and i s p a r t i a l l y  i n h i b i t e d by the presence of KCN and IAA.  Harrison and Harrison [1961]  studied the a b i l i t y o f r a t small i n t e s t i n e ( i n v i t r o ) to concentrate phosphate i n the basal bathing medium. were capable of producing 3 hours of incubation.  They found that t h e i r  preparations  B/A phosphate r a t i o s o f 4.2 - 5.4 to 1 a f t e r They termed the uptake "true transport" of PO^,  but did not characterize the transport mechanism f u r t h e r . An important consideration i n the amount o f Pi appearing basal compartment i s the amount contributed by the t i s s u e .  i n the  The r e c t a l  40 t i s s u e i s capable of c o n t r i b u t i n g a s u b s t a n t i a l amount o f PO^ to the basal compartment, i n the absence o f PO^ i n the bathing medium. c o n t r i b u t i o n by the r e c t a l t i s s u e decreases as the i n i t i a l of PO^ i n the basal bathing medium increases.  This  concentration  P h i l l i p s [1964b, c ] found +  +  that the i n vivo locust rectum decreased i t s rate o f uptake o f Na , K , and C l ~ when the hemolymph concentration of these ions was increased. The observations on PO^ uptake i n t h i s study may r e f l e c t a s i m i l a r mechanism f o r control of phosphate uptake by the l o c u s t rectum.  However,  i t i s also quite possible that PO^ moves by simple d i f f u s i o n from the r e c t a l t i s s u e to the basal compartment.  As the concentration of  phosphate i n the basal bathing medium i s increased, the magnitude of the d i f f u s i o n gradient from the t i s s u e to the basal compartment i s decreased, and t h i s reduced gradient i s observed as a decreased rate of PO^ uptake in the basal compartment.  Although d i f f u s i o n o f f e r s a p l a u s i b l e  explanation of how PO^ moves from the t i s s u e s to the basal compartment, d i f f u s i o n does not explain why poisoned r e c t a , with large amounts of P i in the t i s s u e s , contain l e s s PO^ i n the basal compartment than unpoisoned recta with small amounts of P i i n the t i s s u e s (Table 4) or why accumulat i o n i n the basal compartment i s dependent upon the PO^ concentration i n the a p i c a l bathing medium when the i n i t i a l l e v e l s of PO^ i n the basal compartment are constant (Figure 10).  Harrison and Harrison [1961] also  noticed a t i s s u e c o n t r i b u t i o n to P0^ accumulation  i n the basal compartment  of r a t small i n t e s t i n e , but d i d not i n d i c a t e how much, or how, phosphate moved from the t i s s u e to the basal compartment. I t was of i n t e r e s t to determine whether some o f the phosphate that was found i n the basal compartment was organic or not, because  41 hemolymph of some insects contains 20 - 30 mM/1 organic-P0  A  [Wyatt, 1961].  of a c i d - s o l u b l e  The r e c t a l t i s s u e s perhaps c o n t r i b u t e  organic phosphates to the hemolymph; however, a l l measurable phosphates which were observed to be accumulated i n the present experiments appeared as inorganic P0 . A  32 The t r a c e r experiments i n d i c a t e that  P i s incorporated op  into organic forms by r e c t a l t i s s u e s and i s . a l s o t r a n s f e r r e d as the basal compartment. unlabelled P0  A  transport of P0  The d i l u t i o n of t r a c e r P0  A  Pi to  by a large pool of  i n the t i s s u e means that t r a c e r studies of t r a n s e p i t h e l i a l A  are d i f f i c u l t to i n t e r p r e t .  The e f f e c t s of KCN and IAA, arsenate, and water uptake on accumulation of P0  A  by r e c t a l t i s s u e and w i t h i n the basal compartment,  support the idea of a c a r r i e r on the a p i c a l border of the rectum, but do not exclude a c a r r i e r on the basal border as w e l l . metabolic incorporation of P0  A  KCN and IAA i n h i b i t  i n the r e c t a l t i s s u e s , but do not seem  to i n h i b i t the t i s s u e uptake of Pi from the a p i c a l bathing medium (Table 3 and 4 ) . Arsenate, a competitive i n h i b i t o r f o r P0 decreases the t i s s u e uptake of P0  A  A  uptake systems,  from the a p i c a l bathing medium.  Together, the above two observations seem to i n d i c a t e that a passive c a r r i e r system f o r P0  A  uptake into the t i s s u e e x i s t s on the a p i c a l border  of the rectum.  A  uptake i n the basal compartment i s measured i n  I f P0  the presence of water i n f l u x , the amount of P0 partment does not increase when the P0  A  found i n the basal com-  concentration i n the a p i c a l  bathing medium i s increased from 18 to 61 mM/1 Figure 10).  A  of P0  A  (top curve of  This observation i n d i c a t e s that the saturable c a r r i e r i s  the rate l i m i t i n g step f o r P0  A  uptake i n the rectum; however, where  42 t h i s c a r r i e r i s located on the rectum i s not obvious.  From a t h e o r e t i c a l  point of view i t would seem advantageous f o r the l o c u s t to be able to control the amount of PO^ entering the animal from the environment; hence being able to control the amount of PO^ entering the rectum may  be a case  of having an a p i c a l membrane surface impermeable to phosphate except at s p e c i f i c c a r r i e r s i t e s .  B a l s h i n [1973] found an a c t i v e c a r r i e r f o r  amino acids on the a p i c a l border of the l o c u s t rectum.  PO^ c a r r i e r s have  also been postulated f o r both the a p i c a l and basal border of an Orthopteran's Malpighian tubules [Maddrell, 1971]. 32 The large quantity of by r e c t a l t i s s u e may  Pi incorporated i n t o organic phosphates  i n d i c a t e that phosphorylation i s a key step i n PO^  uptake, by'lowering the a c t i v i t y of Pi i n the t i s s u e . Rothstein [1957] found that glyceraldehyde-dehydrogenase  Goodman and may  in e s t e r i f y i n g PO^ as an a i d to i t s entry into yeast c e l l s .  be important Organelles,  such as mitochondria, which are h e a v i l y concentrated at the a p i c a l  border  of the rectum, perhaps a l s o a i d i n lowering Pi a c t i v i t y i n t h i s part of the c e l l .  Metabolic a c t i v i t y i s important i n determining the amount of  PO^ entering the basal compartment, because r e c t a poisoned with KCN  and  IAA showed a marked decrease i n PO^ accumulation i n the basal compartment (Table 4).  However, KCN and IAA do not g r e a t l y a f f e c t the t o t a l amount  of PO^ entering the r e c t a l t i s s u e from the a p i c a l bathing medium (Table 3). I t i s p o s s i b l e that PO^ uptake by the rectum i s a two step process, c o n s i s t i n g of an entry step from the a p i c a l bathing medium mediated by a f a c i l i t a t e d d i f f u s i o n mechanism, followed by a t r a n s l o c a t i o n step across the c e l l into the basal compartment, mediated by metabolic i n c o r p o r a t i o n  43  When a l l the observations are considered i t i s p o s s i b l e to put together a t e n t a t i v e model f o r PO^ uptake by the i n v i t r o locust rectum. Although t h i s model i s c o n s i s t e n t with the f i n d i n g s of t h i s study, i t i s by no means the only explanation f o r the observations, but i s of value i n so f a r as the experiments i t suggests.  Apical Bathing Medium  Intima  Rectal Tissue  Basal Bathing Medium  S a l i e n t features of the Model: (a)  c a r r i e r that transports PO^ into the rectum by f a c i l i t a t e d d i f f u s i o n located on the a p i c a l border. i n h i b i t e d by arsenate.  This c a r r i e r i s  Entry i s increased by high P 0  4  concentration i n the r e c t a l lumen caused by more r a p i d water reabsorption [Speight, 1967]. (b)  metabolic i n c o r p o r a t i o n of PO^ i n the r e c t a l t i s s u e . step i s i n h i b i t e d by KCN and IAA.  This  44 (c)  PO^ t r a n s f e r from the r e c t a l t i s s u e i n t o the basal compartment i s mediated by e i t h e r a c a r r i e r on the basal membrane or by simple d i f f u s i o n (or both).  Transfer by e i t h e r  mechanism ( c a r r i e r or d i f f u s i o n ) would be influenced by metabolic poisons because the l a t t e r cause large changes i n t i s s u e l e v e l s of inorganic PO^. The f a c i l i t a t e d entry step on the a p i c a l border of a c e l l , followed by metabolic i n c o r p o r a t i o n of the permeant i s a process which f a c i l i t a t e s entry of substrates i n other organisms. studying glucose uptake in. Neurospora d i f f u s i o n entry step f o r glucose.  crassa,  Scarborough [1970],  found a f a c i l i t a t e d  Glucose, upon entry i n t o the c e l l s ,  was phosphorylated and then shunted i n t o the general metabolism cell.  of the  When a non-metabolizable analog (3-0-methyl-D-glucose) was  used  as the substrate f o r the c a r r i e r , the concentration i n the c e l l s e q u i l i b r a t e d with the bathing medium, but d i d not exceed that of the bathing medium (cell/medium glucose concentration r a t i o = 1 ) .  Jain  [1972] terms t h i s type of uptake "loosely coupled energized t r a n s p o r t , " and although i t may not be an accurate d e s c r i p t i o n of P0^ transport i n the l o c u s t rectum, the process seems to be  analogous.  Because of the presence of hormones and other f a c t o r s jm vivo [Mordue, 1969], i n v i t r o systems do not n e c e s s a r i l y a c c u r a t e l y r e f l e c t i n vivo mechanisms, but i t i s s t i l l of value to e x t r a p o l a t e i n v i t r o findings to the whole animal. Values are a v a i l a b l e f o r c a l c u l a t i n g the rate of P0^ s e c r e t i o n by the Malpighian tubles of the l o c u s t .  Speight [1967] found that  45 primary Malpighian f l u i d contains 14.6 ± 5.65 nM/ul (mean ±S.D.) of PO4 ( i n vivo measurement).  Maddrell and Klunsuwan [1973] found that an  i n v i t r o preparation of l o c u s t Malpighian tubules produced a primary f l u i d containing 12 nM/ul of P 0 . A  P h i l l i p s [1964c] found that the -  Malpighian tubules of the l o c u s t secreted a t a rate of 8 ul/hour.  The  estimated Malpighian tubule s e c r e t i o n rates f o r P 0 are 8 x 12 = 96 nM A  of P0 /hour, and 8 x 14.6 = 116.8 nM of P0 /hour. A  A  I f the rate of P 0  A  uptake into the basal compartment by r e c t a incubated i n a p i c a l bathing medium containing 18 mM/1 of P 0 i s taken from Figure 10, uptake values A  of 99.3 ± 7.65 nM of P0 /hour/rectum (water flow across the r e c t a l wall A  prevented), and 80.4 ± 11.2 nM of P0 /hour/rectum (water flow across the A  r e c t a l wall not i n h i b i t e d ) ( v a l u e s are mean ±S.E.) are obtained.  The  values f o r the Malpighian tubule e x c r e t i o n rates and r e c t a l reabsorption rates f o r P 0 correspond q u i t e c l o s e l y . A  A l l of the P 0 excreted by the A  Malpighian tubules can be resorbed by the rectum. The l o c u s t rectum, t h e r e f o r e , has the r e s o r p t i v e c a p a b i l i t y expected of a s i t e responsible f o r P 0 r e g u l a t i o n i n the l o c u s t . A  P0  A  i s r a p i d l y accumulated when hemolymph l e v e l s are low, and i s more slowly accumulated when hemolymph l e v e l s are higher.  The s a t u r a t i o n mechanism  for P 0 uptake would allow excess P 0 to be voided with the feces should A  i n t e r n a l l e v e l s become too high.  A  46  SUMMARY 1.  Net t r a n s f e r of calcium or magnesium across the i n v i t r o l o c u s t rectum was not observed.  2.  Phosphate i s accumulated i n the basal compartment of the i n v i t r o l o c u s t rectum against large concentration d i f f e r e n c e .  3.  The r e c t a l t i s s u e i s capable of c o n t r i b u t i n g a s u b s t a n t i a l amount of inorganic P0  A  to the basal compartment i n the absence of P0  in  A  the basal bathing medium. 4.  Only inorganic phosphate (no organic phosphates) i s accumulated i n the basal compartment.  5.  Water uptake (solvent drag) does not increase the amount of P 0  A  accumulated i n the basal compartment. 6.  The P0 and V  7.  P0  A  A  m a x  uptake f i t s Michaelis-Menton k i n e t i c s with of 52.6 nM/hr/mg D.W.  of 5.0  rectal tissue.  entry into the basal compartment i s i n h i b i t e d by 2 mM  and 2 mM IAA/1.  mM/1  Arsenate i n h i b i t s P0  A  KCN/1  entry from the a p i c a l  bathing medium i n t o the r e c t a l t i s s u e . 8.  A possible mechanism f o r P0  A  uptake by the rectum i s proposed.  47  BIBLIOGRAPHY  BALSHIN, M. and J.E. PHILLIPS, 1971.  A c t i v e absorption of amino acids  i n the rectum of the desert l o c u s t {Schistocerca Nature New Biology.  BALSHIN, M.  1973.  gregaria).  233: 53-55.  Absorption of amino acids i n the rectum of the  desert l o c u s t {Schistocerca  gregaria).  Ph.D. Thesis,  U n i v e r s i t y of B r i t i s h Columbia, Canada.  BERRIDGE, M.J. 1968.  Urine formation by the Malpighian tubules of  Calliphora.  I. Cations. J . Exp. B i o l . 48: 159-174.  BERRIDGE, M.J. 1969.  Urine formation by the Malpighian tubules of  Calliphora.  I I . Anions, J . Exp. B i o l . 50: 15-28.  CLARK, E.W. and R. CRAIG.  1953.  The calcium and magnesium  i n the hemolymph of c e r t a i n i n s e c t s .  content  P h y s i o l . Zool. 26:  101-107.  CLARK, E.W.  1958.  A review of l i t e r a t u r e on calcium and magnesium  in insects.  Ann. Ent. Soc. Am.  51: 142-154.  ERNSTER, L., R. ZETTERSTROM, and 0. LINDBERG.  1950.  A method f o r  the determination of t r a c e r phosphate i n b i o l o g i c a l m a t e r i a l . A Chem. Scand. 4: 942-947.  48  GOH, S.L. 1971. Mechanism o f water and s a l t absorption i n the i n v i t r o l o c u s t rectum.  M. Sc. Thesis, U n i v e r s i t y o f  B r i t i s h Columbia, Canada.  GOMORI, M.D.  1942. A m o d i f i c a t i o n o f the c o l o r i m e t r i c phosphorus  determination f o r use with the p h o t o e l e c t r i c c o l o r i m e t e r . J . Lab. C l i n . Med. 27: 955-960.  GOODMAN, J . and A. ROTHSTEIN.  1957. The a c t i v e t r a n s p o r t o f  phosphate into yeast c e l l s .  J . Gen. P h y s i o l . 40:  915-923.  HARRISON, H.E. and H.C. HARRISON.  1961. I n t e s t i n a l transport o f  phosphate: a c t i o n o f vitamin D, calcium, and potassium. Am. J . P h y s i o l . 190: 1007-1012.  JAIN, M.A. 1972. The bimolecular  lipid  membrane. Van Nostrand  Rheinhold Company, New York.  LEVINSON, C.  1971. Phosphate transport i n E h r l i c h A s c i t e s tumor  c e l l s and the e f f e c t o f arsenate.  J . Cell. Physiol.  79: 73-78.  MADDRELL, S.H.P. 1971. The mechanism o f i n s e c t excretory systems, in "Advances i n Insect Physiology."  Ed. by J.W.L. Beament,  J.E. Treherne and V.B. Wigglesworth, 8: 200-324. Academic Press, London, New York.  49  MADDRELL, S.H.P. and S. KLUNSUWAN.  1973. F l u i d secretion.by i n  v i t r o preparations of the Malpighian tubules o f the desert locust Schistocerca  gregaria.  J . Insect P h y s i o l . 19:  1369-1376.  MARTIN, J.B. and D.M. DOTY.  1949. Determination of inorganic phosphate  Anal. Chem. 21: 965-967.  MORDUE, W.  1969. Hormonal c o n t r o l of Malpighian tubule and r e c t a l function i n the desert l o c u s t , Schistocerca  gregaria.  J.  Insect P h y s i o l . 15: 273-285.  PHILLIPS, J.E.  1964a.  Schistocerca  Rectal absorption i n the desert l o c u s t , gregaria.  F o r s k a l , I. Water. .J. Exp. B i o l .  41: 15-38.  PHILLIPS, J.E. 1964b. Schistocerca  Rectal absorption i n the desert l o c u s t , gregaria.  and Chloride.  PHILLIPS, J.E. 1964c. Schistocerca  F o r s k a l , I I , Sodium, Potassium  J . Exp. B i o l . 41: 39-67.  Rectal absorption i n the desert l o c u s t , gregaria.  the excretory process.  F o r s k a l , I I I . The nature of J . Exp. B i o l . 41: 69-80.  PHILLIPS, J.E. 1965. Retal absorption and renal f u n c t i o n i n i n s e c t s . Trans. Roy. Soc. Can. 3: 237-254.  P h i l l i p s , J.E. and A.A. D o c k r i l l .  1968. Molecular s i e v i n g of  h y d r o p h i l i c molecules by the r e c t a l intima o f the desert locust [Schistocerca  gregaria),  J . Exp. B i o l . 48:  521-552.  RAMSAY, J.A. 1956. Excretion by the Malpighian tubules of the s t i c k i n s e c t , Dixippus  morosus,  (Orthoptera, Phasimidae):  calcium, magnesium, c h l o r i d e , phosphate and hydrogen ions, J . Exp. B i o l . 33: 697-708.  RAMSAY, J.A. 1971. Insect rectum. P h i l . Trans. Roy. Soc. London. 262: 251-260.  ROTHSTEIN, A.  1963. Interactions of arsenate with the phosphate-  transporting system of yeast.  J . Gen. P h y s i o l . 46: 1075-  1085.  SCARBOROUGH, G.A.  1970. Sugar transport i n Neurospora  orassa.  J. B i o l Chem. 245: 1694-1698.  SPEIGHT, D.I. 1967. A c i d i f i c a t i o n of r e c t a l f l u i d i n the l o c u s t Schistooeroa  gregaria.  M. Sc. Thesis, U n i v e r s i t y of  B r i t i s h Columbia, Canada.  51 STEIN, W.D.  1967. The movement of molecules across c e l l membranes.  Academic Press, New York and London.  WYATT, G.R.  1961.  The biochemistry of i n s e c t hemolymph.  Rev. Ent. 6: 75-102.  Ann.  

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