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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 b y EDWARD WILLIAM ANDRUSIAK B.Sc, University of Manitoba, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1974 In presenting th i s thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada 7 ABSTRACT The a b i l i t y of the locust 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 differences. Mg was not accumulated in the basal compartment of the rectal sac even when Mg + + concentration gradients were favourable for net d i f f u s i o n . Phosphate was found to be accumulated by the rectum against a three-fold concentration difference. The rectal tissue incorporated inorganic phosphate into organic forms, but phosphorus transferred 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 saturation kine t i c s . The resorption of water by the rectum did not increase (by solute drag) the amount of PO^ accumulated in the basal compartment, except at very high PO^ concentrations i n the apical bathing medium. Metabolic poisons such as KCN and IAA inhibited PO^ accumulation i n the basal compartment but did not i n h i b i t PO^ entry into the tissue from the apical bathing medium. Arsenate, a competitive i n h i b i t o r of PO^ uptake i n other systems, inhibited PO^ entry into the tissue. A mechanism for PCL uptake into the tissue i s proposed. 11 i 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 in Media and Tissues 11 Electropotential Differences Across the Rectal Sac . . . . . 12 Treatment of Results 14 RESULTS 16 Viability of the In Vitro Rectal Preparation 16 Test for Accumulation of Ca , Mg , and PO^  17 Net Ca Movement 1" Net Mg++ Accumulation 19 Net PO^  Accumulation 22 Tissue Contribution to Uptake of PO. in the Basal Compartment 25 Type of PO^  (Inorganic and/or Organic) in the Basal Compartment 28 Effect of Water Uptake on PO. Accumulation in the Basal Compartment 29 Kinetics 29 Page Inhibition of PO^ Transfer 3 2 Further Characterization of the System 34 DISCUSSION . . . . . 3 8 SUMMARY 4 6 V LIST OF TABLES Table ; Page I. Composition of Bathing Media 10 I I . The Net Movement of C a + + into the Rectal Tissue and into the Basal Bathing Medium, Over 3 Hours of Incubation in Media Containing Various Amounts of C a + + 2 1 I I I . Net 3 2 P Uptake in Either Control or Poisoned Tissue, After 6 Hours of Incubation . 33 IV. Various Fractions of PO4, Determined by Chemical Analysis and Radiotracer Estimation ( 3 2 P ) , in In-Cubated and Unincubated Recta 35 v i LIST OF FIGURES Figure Page 1. Diagram of experimental set-up to study rectal absorption i j i v i t r o ~ 2. Apparatus used for measurement of trans rectal potentials 1 3 3. Trans rectal potential differences (apical side positive) across everted rectal sacs with time after preparation 15 4. The net rate of water movement across the rectal wall in the absence and presence of an osmotic gradient ^ The net movement of C a + + and water across the rectal w a l l , after 3 hours of incubation in Ringer containing various amounts of C a + + . . 20 6. The net movement of Mg + + and l-LO across the rectal wall at various Mg + + concentrations in the apical bathing medium, with and without net water move-ment across the rectal wall . 23 7. The total amount of Mg + + in rectal tissue a f t e r 4 hours of incubation, with and without sucrose present i n the apical bathing medium 24 8. The basal: apical (B/A) concentration r a t i o of P0^ across in v i t r o rectal sacs with time 26 9. The f i n a l P 0 ^ r a t i o (B/A) and the net movement of water across the rectal sacs (after 6 hour of i n -cubation) when the P 0 * concentration of the bathing media was varied 27 1 0 . The net rate of P O 4 appearance in the basal com-partment and the net water movement across the rectal sac for a range of PO4 concentrations in the apical bathing medium 30 1 1 . Double reciprocal plot of the net rate of PO4 entry into the basal compartment as a function of the P O 4 concentration in the apical bathing medium 31 v i i LIST OF ABBREVIATIONS B/A basal/apical side u l m i c r o l i t e r ml mi 1 1 i 1 i ter nM nanomoles mM millimoles P.E-. polyethylene tubing mOsm milliosmoles mV m i l l i v o l t s hr hour mg milligram D.W. dry weight S.E. standard error P.D. potential difference K t substrate concentration for half-maximal unidirectional f l u x V max maximal rate of unidirectional f l u x ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. J.E. P h i l l i p s , for his guidance and helpful discussions during the preparation of this thesis. I am also grateful to Ms. Joan Meredith and Dr. M. Balshin for th e i r assistance. 1 INTRODUCTION The role of ionic regulation, osmotic regulation, and metabolic waste removal f a l l s mainly upon the Malpighian tubule-rectal 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 rectal resorption i s analogous to the glomerular f i l t r a t i o n and tubular resorption of the vertebrate kidney [ P h i l l i p s , 1965]. The Malpighian tubules produce a primary f l u i d that i s nearly iso-osmotic with, but not necessarily 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 driving force responsible for the production of primary f l u i d i s the + + active 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 into the tubule lumen [Maddrell, 1971], Small organic molecules (sugars, some amino acids) may be brought into the lumen by solvent-solute or solute-solute drag, while large organic molecules (organic acids, nitrogenous wastes) may be a c t i v e l y secreted [Maddrell, 1971]. The rate of f l u i d secretion by the Malpighian tubules of most insects seems to be d i r e c t l y related to the K + concentration of the basal bathing medium [Ramsay, 1956; Berridge, 1968; Maddrell and + + Klunsuwan, 1973]. K and Na produce the highest rate of secretion, 2 but anions also influence secretion. The rate of secretion i s inversely related to the hydrated radius of the secreted anion [Berridge, 1969]. Phosphate is an exception since i t can support a high rate of secretion completely disproportionate to i t s hydrated radius [Berridge, 1969]. The primary f l u i d , containing both metabolic wastes and physiologically important solutes, flows out of the Malpighian tubules into the midgut-hindgut junction where i t then flows to the rectum via the anterior part of the hindgut. The concentration of the con-stituents of the primary f l u i d are altered to a minor degree by the d i s t a l portions of the Malpighian tubules in some insects and by the anterior portion of the hindgut of others'. The bulk of the resorption, however, occurs in 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 . Many of the hemolymph constituents are a c t i v e l y resorbed [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 apical side by a chitinous 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 results in the accumulation of metabolic wastes in the rectum due to the action of the intima. Since the Malpighian tubule-rectal complex also controls osmotic pressure and ionic concentrations of the hemolymph, the rate of Malpighian tubule secretion i s closely correlated with rectal reabsorption rates [ P h i l l i p s , 1964c]. P h i l l i p s [1964 a, b, c] studied Na , K , CI , and water secretion and subsequent resorption in the 3 locust and found that i f the balance of ions in the hemolymph was perturbed, the r e l a t i v e secretion 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 in excess w i l l accumulate in the rectum and ions in short supply w i l l be completely reabsorbed. Water was found to be reabsorbed rapidly under a l l experimental s i t u a t i o n s . Na , K , CI , and water have been the focus of attention in ++ ++ most of the investigations to date. PO^, Mg , and Ca have been less intensely studied, and then only from the point of view of secretion by the Malpighian tubules. Since PO^ can af f e c t the rate of production of primary Mal-pighian f l u i d [Maddrell, and Klunsuwan, 1973] and i s also a metabolic regulator [Levinson, 1971], i t would seem that thellocust should be able to regulate the secretion and reabsorption of PO^ to some extent. Speight [1967] and Maddrell and Klunsuwan [1973] found that PO^ was more concentrated in the primary Malpighian f l u i d than in the hemolymph or in the external bathing medium. Maddrell and Klunsuwan [1973] found that in v i t r o preparations of locust Malpighian tubules were capable of producing a lumen to hemolymph PO^ r a t i o of 1.71 to 1.0. Berridge [1969] found that 1.0 mM/1 of arsenate, when added to incubation media containing PO^ as the most abundant anion, greatly 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 also a competitive i n h i b i t o r for 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 translocation 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 divalent cations Mg and Ca do not seem to be a c t i v e l y secreted by the Malpighian tubules. Ramsay [1956], while working with in v i t r o preparations of the Malpighian tubules of the s t i c k insect, found Mg and Ca to be excreted at 1/10 to 1/5 of the concentration ++ ++ of the two ions in the bathing medium. Concentrations of Mg and Ca above 10 mM/1 in the bathing media inhibited the rate of f l u i d secretion by the Malpighian tubules [Berridge, 1968]. The hemolymph, however, contains C a + + and Mg + + ions at concentrations more than fo r t y times greater than in mammalian blood [Clark and Craig, 1953]. The same authors concluded that much of the C a + + and Mg + + i s not phys i o l o g i c a l l y active in the hemolymph and i s probably sequestered by proteins and other organic compounds. Calcium carbonate i s found i n the Malpighian tubules of many insects [Clark, 1958], and i s usually eliminated as a suspension of granules. The high concentrations of p o t e n t i a l l y physio-l o g i c a l l y active Mg and Ca in the hemolymph may not necessitate the reclamation of these ions by the rectum, hence excretion as precipitates. The present study was undertaken with two main objectives in mind. These were: 1. To determine i f Ca , Mg , and PO^ are reabsorbed in the locust rectum, as are monovalent ions,, and to compare rates of resorption with rates of secretion 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 in vivo, was used because of i t s greater potential for 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 , active transport) of net absorption of any of these ions. After doing a b r i e f study on the uptake,of the three ions, 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 thesis i s con-cerned with the mechanism and l o c a l i z a t i o n of the uptake system f o r P0„ in the locust rectum in v i t r o . 6 MATERIALS AND METHODS Animals Adult male and female locusts {Schistooeroa gregaria Forskal), from a colony maintained at 28°C and 60% r e l a t i v e humidity, and fed a diet of bran and lettuce, were used i n a l l experiments. Locusts were used from one to f i v e weeks after t h e i r f i n a l molt. Locusts were starved for 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 rectal sacs were prepared according to the method of Goh [1971]. The locust, 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 dissecting microscope, was anaesthetiz-ed with a mixture of CO2 and ether. A U-shaped, do r s a l - l a t e r a l i n c i s i o n was made from the f i f t h to the seventh abdominal segment. The resulting flap of c u t i c l e was pinned back revealing the rectum with associated tracheae and f a t bodies. These structures were c a r e f u l l y dissected away from the rectum. 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 flared end was ju s t anterior to the rectal pads. A liga t u r e of clean human hair was used to secure the rectum to the fl a r e d end of the cannula. The hindgut anterior 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 rectal pads emerged. The everted rectum was then severed from the body of the locust. The external surface of the cannulated rectum was rinsed in 10 ml of Ringer solution (Table 1), and the internal surface was flushed with about 0.25 ml of Ringer solution injected through P.E. 10 tubing attached to a syringe. This procedure removed faecal material or hemolymph adhering to the rectum. Another hair was used to lig 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 in the sac. The head of a bent insect pin was inserted into the end of the cannula, thus forming a hook by which the rectal 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 solution were injected into the sac with a 'Hamilton Syringe 1 and the sac was reweighed to obtain the weight at zero-time of incubation. Occasionally a few recta were checked for leakage by leaving them overnight i n an amaranth solution. 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 in a sac would indicate damage to the rectum. No leakage was observed. Incubation Procedure The rectal 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 in 3 - 25 ml of apical (external) bathing medium. Twenty ul of media were used to bath the basal (internal) surface of the rectal preparation. For experiments of short duration, sacs were pre-incubated for one hour to l e t them achieve a steady state [Goh, 1971; Balshin, 1973]. At the end of one hour the contents of the sacs were removed and 20 ul of fresh basal bathing media were added. The sac was then reweighed and transferred to'fresh apical bathing medium. To determine i f any water was taken up by swelling of the rectal tissue, weights of the empty sacs at zero and f i n a l time (t) were compared. I f any changes occurred, the difference was either added or subtracted from the amount of water taken up i n the basal compartment. In this way i t was possible to determine i f net water transfer across the rectal sac occurred during the experiment. In a l l experiments, the weighing procedure consisted of removing the sac from the bathing media, blotting i t dry (with Whatman No. 1 f i l t e r paper), then weighing i t on a 200 mg 'August-Sauter' torsion balance. " 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 apical bathing medium in most experiments. The concentration of the ion under study was altered in some experiments;" such changes are noted in describing the r e s u l t s . The two Ringers were used in order to roughly approximate in vivo 9 Figure 1 Diagram of experimental set-up to study rectal absorption in v i t r o . The everted rectal sac, containing 20 ul of experimental media in the basal compartment was incubated i n 3-5 ml of apical medium. Mixing and aeration was achieved by bubbling 95% 0o and 5% C0 ? through apical medium. 9a 95% O2 5 % C 0 2 Insect pin Cannula Incubation Vessel Everted Rectal Sac Basal 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 • 6H20 0.73 0.73 CaCl 2 • 2H20 0.315 0.315 Dextrose 3.0 3.0 NaHC03 0.18 0.18 NaH 2P0 4 • 1H20 0.84 0.84 Sucrose* 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 internal media was set at pH 7.0 [Speight, 1967; P h i l l i p s , 1964a]. A 'Radiometer Model 25' was used to make a l l pH readings. The pH of the apical bathing (external) medium during the course of experiments did not vary from the zero-time reading. Measurement of Ion Concentrations in Media and Tissue Ten ul aliquots were removed from the apical and basal bathing media and were analyzed for the desired ion. 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 for tissue analysis, because i t did not dephosphorylate l a b i l e organic phosphates as readily as did the method of Gomori [Martin and Doty, 1949]. A 'Spectronic 20' was used for the colorimetric determinations. Samples for Mg + + 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 1 l i q u i d s c i n t i l l a t i o n counter. 1-3 ul samples were collected with 'Drummond Microcaps' micropipettes. These samples were put into 10 ml •of either Bray's solution [Bray, 1960] or Aquasol (New England Nuclear) for counting. 32 Fluid samples (1-3 ul) containing P were placed on planchets, dried and then counted on a 'Nuclear Chicago Model 1042' automatic planchet counter. 12 32 Tissues to be analyzed f o r P were f i r s t rinsed with i s o -osmotic mannitol and were blotted dry with f i l t e r paper. The recta l tissue was then spread out evenly on a planchet and was dried slowly under an infra-red lamp. The planchet with the dried tissue was counted on a 'Nuclear Chicago Model 1042' planchet counter. 45 Tissues analyzed for Ca content were rinsed with i s o -osmotic mannitol, blotted, put into weighed platinum boats, and were weighed. The tissues were dried for 48 hours at 80°C i n a drying oven. The dry weights were found, and then the tissues were dry ashed in a muffle furnace at 460°C for 24 hours [ P h i l l i p s , 1964b]. The ash was dissolved in 1 ml of d i s t i l l e d water and 3-25 ul samples were taken with Lang-Levy pipettes. These samples were put into 10 ml of 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 for Mg were ashed by the same pro-++ + cedure as for Ca . The ash was dissolved in 3 ml of 0.75% Na -EDTA and the Mg + + content was read on the flame spectrophotometer ('Techtron 120AA') in the atomic absorption mode of operation. Inorganic phosphate (Pi) was extracted from a tissue homogenate with ice cold 10% TCA. The precipitate was centrifuged, washed with more ice cold 10% TCA, then centrifuged again. The supernatant was decanted and analyzed for Pi by the method of Ernster et a]_. [1950]. Electropotential Differences Across the Rectal Sac In order to determine the direc t i o n and the magnitude of the potential difference (P.D.) across the rectal sac, the apparatus shown in Figure 2 was used . A 'Keithly Model 602' electrometer was used 13 Figure 2 Apparatus used for measurement of transrectal potentials. The asymmetry potential was obtained by immersing the the basal end of the KC1 bridge into the apical medium. KEITHLY MODEL 602 ELECTROMETER 9 5 % 0 2 5 % C 0 2 Calomel Electrode 3M/L KCl Bridge Basal End of KCl Bridge Everted Rectal Sac Apical Bathing Medium 3M/L KCl — 14 to measure potential differences. 'Radiometer' calomel electrodes i n series with a 3 M/l KCl-agar bridge made up in P.E. 10 tubing completed the c i r c u i t . The asymmetry potential difference was found by inserting the agar bridge, shown on the basal side, into the vessel containing the apical bathing medium. The asymmetry potential was subtracted from the measured trans-rectal potential differences. Treatment of Results The Student's t-test was used to s t a t i s t i c a l l y test for significance of the observed differences. Unless the probability level 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 probability less than or equal to 0.05. 15 Figure 3 Trans-rectal potential differences (apical side positive) across everted rectal sacs with time after preparation. Na-Ringer was present in both compartments (apical and basal) and 420 mOsm/1 of sucrose was added to the apical medium to prevent water movement. The v e r t i c a l bars represent ±S.E. of the mean (7 preparations). o m to X o 0 C/) POTENTIAL DIFFERENCE (mV) o 0 0 o 4S o o CD O ^4 O \ \ \ 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 of in v i t r o preparation used in this study. His results were comparable to those observed in vivo + + by P h i l l i p s [1964b] with respect to the active transport 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 active transport of amino acids. He used the electropotential difference across the rectal w a l l , and the uptake rate of H20 to assess the s t a b i l i t y of the preparation with time. A steady state condition was attained after one hour of preincubation and was maintained for at least 6 hours. The preparations used in this study exhibited the same degree of s t a b i l i t y with regard to P.D. (Figure 3) and H20 uptake rate (Figure 4) for at least 6 hours, the longest period of time during which experiments were conducted. Water movement into the rectal sac was blocked or decreased in some experiments by the addition of 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 apical bathing medium. Na +-Ringer requires 420 mOsm/l of sucrose in the apical bathing medium to block H20 movement [Balshin, 1973], but K +-Ringer requires 586 mOsm/l of sucrose to block H20 movement (Figure 4). This seems to indicate that the preparation can remove H20 more e f f e c t i v e l y from a K +-Ringer than from a Ma -Ringer. Water i s also absorbed at 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, Balshin, 1973] i n the absence of an osmotic difference across the rectal sac. The i n i t i a l potential difference 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 positive, for the next 5 hours. The observed P.D. was of the same po 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. of the magnitude observed i n v i t r o under certain conditions. The s t a b i l i t y and magnitude of the observed P.D. (50-60 mV, lumen positive) agrees closely with that measured i n v i t r o by Balshin [1973] during the 5 hour steady-state period. ++ ++ Test for Accumulation of Ca , Mg , and P0^ To see i f these inorganic ions accumulated across the rectal w a l l , equal concentrations of the ion were placed on both sides of the everted rectal sacs at zero-time. Accumulation of ions with time would indicate some driving force other than concentration difference. Factors such as solvent drag and P.D. could then be examined to see i f they could account for the accumulation. If solvent flow i s prevented and the P.D. can not account for an observed accumulation, active transport might then be postulated. ++ ++ The concentrations of free (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 for the s t i c k insect; Ramsey, 1956]. 18 Figure 4 The net rate of water movement across the rectal wall i n the absence and presence of an osmotic gradient. The baching medium i n the apical 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 apical medium. Vertical bars represent ±S.E. of the mean (4 preparations). 19 The Ringer concentrations (Ca 2.14 mM/1, Mg 3.59 mM/1) of these ions were therefore considered appropriate to test for net transfer. Net C a + + Movement Basal to apical concentration ratios (B/A) of C a + + did not vary from unity after 3 hours of incubation i n the absence of water movement (Figure 5). Experiments were conducted at two other media ++ concentrations (0.2 and 0.02 mM/1 of Ca ) but again B/A ratios did not exceed unity, indicating only a minimum of Ca movement (Figure 5). When 4 5Ca was placed i n i t i a l l y only in the apical medium (unidirectional flux experiment), a small amount (0.92 ±0.2 nm/mg D.W. rectal tissue) of C a + + was observed in the basal medium after 3 hours of incubation, indicating that the rectal wall i s only s l i g h t l y permeable to Ca from the apical side (Table 2). When the recta are incubated i n 0.2 mM/1 ++ 45 of Ca , with tracer on both sides, 4 times more Ca enters the rectal tissue than under sim i l a r conditions when tracer i s placed only on the apical side. This indicates that C a + + enters the rectum from the basal side much more rapidly than from the apical side (Table 2). Net Mg + + Accumulation In these experiments the Mg + + concentration i n the basal bathing medium was i n i t i a l l y 3.6 mM/1 while the Mg + + concentration in the apical bathing medium was varied over a 100-fold range. The B/A concentration ratios were calculated from the average hourly rates of 20 Figure 5 The net movement of Ca and water across the rectal 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 apical bathing medium (with 586 mOsm/1 of sucrose), and Na -Ringer was present as the basal bathing medium. At zero-time the C a + + concen-trations were the same in both the apical and basal bathing media (0.02, 0.2, or 2.0 mM/1 of C a + + ) . Prepar-ations were pre-incubated for one hour in the experimen-t 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 0 •7 C "1 S J J *2r 0 < UJQ-2 3 - 2 002 0-2 20 INITIAL [Ca++] IN A P I C A L B A T H I N G MEDIUM(mM/L) 21 TABLE 2 The Net Movement of C a + + into 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 apical 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 that of the apical bathing medium, was the basal bathing medium. 586 mOsm/l of sucrose was added to the apical bathing medium to prevent water movement. Recta were pre-incubated for one hour i n the experimental media. The results are expressed as the mean ±S.E. (at least 5 preparations). I n i t i a l Apical and Basal Net Accumulation of C a + + (nM/mg D.W. Rectal C a + + Concentration Tissue) .  ("W 1) In Basal Compartment In Rectal Tissue 0.02 0.2 2.0 * 0.2 (unidirectional flux) 0.04 ± 0.02 1.08 ± 0.6 •1.5 ± 1.0 0.9 ± 0.2 0.5 ± 0.1 6.6 ±1.1 17.9 ± 3.5 1.4 ± 0.1 unidirectional f l u x -- 0.2 mM/1 of C a + + was present i n the apical and basal bathing medium, but tracer ( 4 oCa) was present only in the apical bathing medium. 22 net uptake (Figure 6). Since these rates were so small, the B/A rat i o barely exceeded unity, indicating v i r t u a l l y no basal Mg + + accumulation. Two other observations of interest are evident from Figure 6. F i r s t l y , the i n f l u x of water has no s i g n i f i c a n t effect on ++ ++ the Mg uptake rate except at 36 mM/1 of Mg in the apical bathing medium. Secondly, a tenfold apical to basal concentration difference +4* has no s i g n i f i c a n t effect on the uptake of Mg in the basal compartment when water movement i s prevented. Figure 7 indicates that the rectal tissue content of Mg + + ++ i s r e l a t i v e l y independent of Mg concentration in the apical bathing medium. These measurements were made at the end of four hours of incubation. Freshly extirpated, recta contain 4.8 ± 1.5 nm Mg++/mg D.W. rectal tissue. Net PO4 Accumulation Speight [1967] measured the PO^ concentration of hindgut f l u i d (made up mostly of Malpighian tubule f l u i d ) , and deproteinized hemolymph of locusts. She found conc^ja^fcrtions of 14.6 ± 5.6 and 6.2 ± 1.3 mM/1 of PO4 (mean ±S.D.) respectively. Rectal concentration of P 0 4 was 42 mM/1. With these in vivo PO^ concentrations in mind, rectal pre-parations were incubated in Ringers containing either 4, 12 or 42 mM/1 of PO^ in both apical and basal bathing media. In Figures 8 and 9 the change in PO^ concentration ratios (Basal/apical side; B/A) i s plotted against time, and against PCL concentration i n the apical 23 Figure 6 The net movement of Mg + + and H^O across the rectal wall at various Mg + + concentrations in the apical bathing medium, with and without net water movement across the rectal w a l l . K -Ringer with various Mg concentrations was present as the apical 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 Mg + + concentration in the basal compartment (3.6 mM/1) was the same in a l l experi-ments. The rates are an average of hourly rate measurements taken over 3 hours. Preparations were preincubated for one hour in the experimental media. The v e r t i c a l bars represent ±S.E. of the mean (4 preparations). 23a L J J < C O C O Ui O lli 2 6 0 &50 ^ 4 0 < o r Q30 ov E20 10 0 -10 E c u j ^ 1 5 ! | 10 U- LU c o v : 3 -5 Oh 0 0 - 1 0 20 30 40 [Mg*4] IN APICAL BATHING MEDIUM(mM/l) 24 Figure 7 The total amount of Mg + i n rectal tissue 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 apical bathing medium, with 586 mOsm/1 of sucrose ( 0 ) or with-out 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 Mg + + in a l l experiments. The v e r t i c a l bars represent ±S.E. of the mean (4 preparations). M g + * C O N T E N " (nm/mg D.W. R OF RECTA C T A L T I S S U E ) O o 4N O —I Y 25 bathing medium, respectively. The highest B/A r a t i o (ca. 3) was obtained in 2 hours and was maintained for at least 4 more hours i n an experiment where 4 mM/1 PO^ was placed i n both apical and basal bathing media at zero-time. Preparations incubated in both 12 and 42 mM/1 of PO^ showed a gradual increase in 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 tiss u e , preparations were incubated for 6 hours in PO^-free medium with 586 mOsm/l of sucrose in the apical bathing medium. During this period 114.8 ± 14.7 nm PO^ rectum (mean ±S.E.) accumulated in the basal compartment. This was almost as much as the tota l accumulation of PO^ (139.0 ± 25.3 nm/retum) at 6 hours i n the basal compartment when the recta were incubated in apical 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 in the apical bathing medium, a total of 60 nM/rectum of P0^ accumulated in the basal compartment af t e r 6 hours of incubation. *It seems, that when the basal bathing medium contains l i t t l e or no P0^, the rectal tissue can contribute substantial amounts of P0^ to the basal compartment. Since the P0^ accumulation in the basal compartment increases with increasing P0^ concentration in the apical bathing medium, when the PO^ concentration i n the basal 26 Figure 8 The basal: apical (B/A) concentration r a t i o of P0 4 across in v i t r o rectal sacs with time. K -Ringer was the apical bathing medium, and Na -Ringer was the basal bathing medium. The apical and basal bathing media both contained i n i t i a l l y either 4( © ), 12 ( © ), or 42. ( A ) mM/1 of P0 4. The apical 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. of the mean (4 preparations). -0 26a 27 Figure 9 The f i n a l PO4 r a t i o (B/A) and the net movement of water across the rectal sacs (after 6 hours of incubation) when the PO^ concentration of the bathing media was varied. + + K -Ringer was the apical bathing medium and Na -Ringer was the basal bathing medium. The apical and basal bathing media both contained i n i t i a l l y either 4, 12, or 42 mM/1 of PO4. The apical bathing medium contained 586 mOsm/l of sucrose. The v e r t i c a l bars represent ±S.E. of the mean (4 preparations). 1 D_ 0 c r + 4 L U " T , ^ v : 0 < LUCL - 4 0 r 10, 2 0 30 4 0 45 INITIAL[P04] IN APICAL AND B A S A L B A T H I N G M E D I A ( m M / D 28 compartment i s i n i t i a l l y held constant, at least part of the accumu-l 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 It was of interest to see i f the PO^ accumulating in the basal compartment was a l l inorganic. The method of Ernster et aj_. [1950] was used to measure the Pi present i n an aliquot drawn from the basal bathing medium. Another aliquot from the same medium was hydrolyzed with hot acid, thus cleaving PO^ from a c i d - l a b i l e organic phosphates. The PO^ was then measured by the method of Ernster et al_. [1950]. The amount of PO^ did not increase over the amount of inorganic phosphate measured in the f i r s t aliquot; 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 test was conducted when P was used to estimate the rate of accumulation of PO^ in 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 Pi) from an aliquot taken from a sample of basal bathing 32 medium. The same aliquot with the Pi removed was then counted on a clanchet counter. Only the background reading was observed, indicating 32 that no P was present in an organic form. 29 Effect of Water Uptake on PO^ Accumulation i n the Basal Medium To test the effect of r^O uptake on the accumulation of PO^ in the basal compartment, recta were incubated i n Ringer with no sucrose present, hence no osmotic difference existed between the apical and basal compartments. No difference in the rate 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 in the apical bathing medium was low (Figure 10). However, at the highest PO^ concentration in the apical medium (61 mM/1), H20 uptake has a s i g n i f i c a n t (p < 0.001) effect on the rate of PO^ transfer into the basal compartment. Kinetics Saturation kinetics are obtained when the net rate of PO^ uptake in the basal compartment i s plotted against a 100~fold external concentration range (0.6 - 61.0 mM/1) of PO^ (Figure 10). The zero-time concentration of PO^ in the basal compartment was 6.0 mM/1 of PO^ in a l l experiments. The data f i t Michealis-Menton kinetics with K t of 5.0 mM/1 and V of 52.6 nM/hr/mg D.W. rectal tissue (Figure 11). max Saturation kinetics i s a phenomenom associated with c a r r i e r mediated processes. If PO^ was di f f u s i n g into the basal compartment, a linear relationship (Fick's Law) should occur when rate i s plotted against the PO^ concentration in the apical bathing medium. This was not the case. Stein [1967] considers saturation as " r e l a t i v e l y strong" evidence for carrier-mediated systems. 30 Figure 1 0 The net rate of P 0 ^ appearance in the basal compartment and the net water movement across the rectal sac for a range of PO^ concentrations in the apical bathing medium. K +-Ringer with varied P 0 » concentrations was the apical bathing medium, with 420 mOsm/l of 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 of P O 4 appearance in th§ basal compartment was measured over a period of 1 hour. The v e r t i c a l bars represent ±S.E. of the mean (at least 4 preparations). 30a ~6Qr LLJ 3 o )50 ^ £ 2 5 r L L V o < 10 0 0-0 0 10 20 30 60 [PO4] IN APICAL BATHING MEDIA(mM/l) 31 Figure 11 Double reciprocal plot of the net rate of PO^ entry into the basal compartment as a function of the PO^ concentration in the apical bathing medium. This plot 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 apical bathing medium. 31a 32 Inhibition of Phosphate Transfer Another test for c a r r i e r mediated systems involves uptake rate measurements in the presence of substrate analogs. Analogs compete with a natural substrate for 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 for the active PO^ uptake system i n yeast c e l l s . Berridge [1969], found that arsenate inhibited PO^ driven secretion by the Malpighian tubules of an insect, and postulated that the arsenate was competing (with PO^) for 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 of PO^ and 8 mM/1 of AsO^ i n the apical and basal bathing media. 586 mOsm/l of sucrose was present i n the apical bathing medium to prevent h^ O movement. 32 The tota l amount of P that entered into the tissue over a 6 hour period was measured after incubation i n the absence and in the presence of AsO^. Twice as much PO^ accumulated i n the control tissue (p < 0.01) indicating AsO^ i n h i b i t s P0^ uptake (Table 3). However, as well as being a competitive i n h i b i t o r for PO^ uptake systems, AsO^ i s also a metabolic poison; so at this point i t was not possible 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 of potassium cyanide and 2 mM/1 of iodoacetic acid with 5 mM/1 of PIPES buffer (piperazine-N, N-bis (2-ethane sulfonic acid)" monosodium monohydrate) and 50 mOsm/l of sucrose in the apical bathing medium at pH 6.6 was used to abolish water uptake in the recta [Balshin and P h i l l i p s , 1971], The control media 33 TABLE 3 32 Net P Uptake i n Either Control or Poisoned Tissue, After 6 Hours of Incubation In experiment 1, K +-Ringer with 586 mOsm/1 of sucrose, 8 mM/1 of 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 for the control except for the absence of ASO4. In experiment 2, Na +-Ringer with 5 mM/1 of PIPES buffer, and 8 mM/1 of P0» was present as the apical and basal bathing medium (control). The apical bathing medium contained 420 mOsm/1 of sucrose. The experimental bathing media.were the same as the control bathing media with the addition of 2 mM/1 of KCN and 2 mM/1 of IAA to both bathing media. The apical bathipg medium (pH 6.6) contained 50 mOsm/1 of sucrose. The results are expressed as the mean ±S.E (6 preparations). Experiment Apical Bathing Medium Sucrose in Apical Bathing Medium (mOsm/1) Tissue Uptake of 32p (nM PO4/rectum) Net H?0 Move-ment ( u l / rectum) 1 K +-Ringer 586 107.3 ± 8.6 +1.4 ± 3.4 1 K +-Ringer plus 8mM/l of As0 4 .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 50 47.4 ± 6.7 -0.2 ± 2.4 and IAA 34 contained 420 mOsm/l of sucrose i n the apical bathing medium to abolish h^ O uptake. After 6 hours of incubation (no pre-incubation), 32 no s t a t i s t i c a l difference (p > 0.1) i n tissue P uptake could be observed between the control and test preparations. Seemingly, P0^ entry into the rectal tissue from the apical bathing medium i s not affected by the presence of KCN and IAA i n the apical bathing medium. Since the P accumulation was measured i n the ti s s u e , the apical membrane i s the only barrier between the tissue and the apical bathing medium. Therefore, unless AsO^ was in 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 inh i b i t o r s KCN and IAA may indicate that the location of the c a r r i e r i s in the apical membrane. Further Characterization of the System The amount of P0^ taken up into the basal compartment, when 32 measured chemically, exceeded that estimated by P uptake by about a factor of 4 (Table 4). This observation prompted an investigation into the tissue 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 freshly extirpated unincubated recta were determined using the method of Ernster frt aj_. [1950]. Another group of recta were then incubated in Na-Ringer with 5 mM/1 of PIPES buffer at pH 6.6. This modified Na-Ringer was 32 the apical and basal bathing medium with the addition of P to the 35 TABLE 4 Various Fractions of PC1*, Determined by Chemical Analysis and Radiotracer Estimation ( P), i n Incubated and Unincubated Recta Recta were incubated for 6 hours. Na -Ringer with 5 mM/1 of PIPES buffer and 8 mM/1 of PO. was the incubation medium i n both the apical and basal compartments for control tissue. In addition, the apical bathing medium (pH 6.6) contained 420 mOsm/1 of sucrose to prevent net water movement. The incubation media for the poisoned incubated tissues were the same as for the control tissues with the addition of 2 mM/1 of KCN and 2 mM/1 of IAA to both bathing compartments. The incubation medium on the apical side (pH'6.6) contained 50 mOsm/1 of sucrose. Results are expressed as the mean ±S.E. (6-12 preparations). Method of PO4 Determina-tion Treatment of Recta Inorganic P0 4(Pi) (nM/rectum) Acid Labile Organic-PO4 (nM/ rectum) Acid Labile Organic-PO4 plus Pi (nM/ rectum) Non-Acid Labile Organic-P0 4 (nM/ rectum) Net Basal Uptake of P0 4 (nM/ rectum) 3 2 P Esti-mation urn ncu-bated incuba-ted con-t r o l 20.5±2.1 45.6 66.H6.9 16.2±0.8 24.0±2.8 incuba-ted poisoned 44.8±6.9 7.8+1.1 29.8±3.1 unincu- 52.2±4.5 66.0 118.2± bated 5.8 Chemical incuba- 115.2± 149.0 264.2± 94.9±8.1 Analysis ted con- 6.1 11.0 t r o l incuba- 173.2± 65.0±4.4 ted 11.7 poisoned 36 apical bathing medium only. After being incubated for 6 hour, the tissues were analyzed for 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 in both Pi and acid l a b i l e organic-PO^ over the unincubated tissues. 32 The values derived for P i n f l u x indicate that roughly three-quarters of the PO^ taken up by the rectal tissue (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 for about one-third of the increase in chemically determined Pi over the amount obtained from unincubated recta. The additional amount may occur as a by-product of energy metabolism, and possibly as a release of previously sequestered unlabelled PO^. If preparations are incubated in the presence of 2 mM/1 of KCN and 2 mM/1 of IAA, the chemically determined tissue Pi increases more than 3 fold (173.2 ± 11.7 nM/rectum) over the unincubated tis s u e , and about 1.5 times (115.2 ± 6.08 nM/rectum) over the amount of Pi in the 32 control tissue. 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) in the poisoned tissue i s about one-sixth of the amount of organic-PO^ in the control tissue (45.6 nM/rectum), indicating that the metabolic i n h i b i t o r s have interferred with the metabolic incorporation of P0^ into organic forms, but have not interferred with the entry of P0^ into the tissue. The chemically determined amount of P0^ in the basal compartment was s i g n i f i c a n t l y greater for the control recta (94.9 ± 8.1 nM/rectum) 32 than for the poisoned recta (65.0 ± 4.4 nM/rectum). However, the P 37 estimations of the amount of PO^ accumulated in the basal compartments of poisoned recta were almost identi c a l with the estimations of the 32 amount of PO^ accumulated by the control recta (also determined by P estimation). This observation seems to implicate metabolism in the accumulation of PO^ in 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 in the ti s s u e , hence i t is not surprising that chemical and isotopic estimations of PO^ accumula-tion in the basal compartment do 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 possibly a s i t e of Mg , Ca , and PO^ ++ 4*4" reabsorption and hence regulation. Ca and Mg (at low concentrations) do" not appear to be transported by the in v i t r o rectum. PO^ i s accumulated in the basal compartment against a sizeable electrochemical gradient. These findings are perhaps not surprising when observations from the l i t e r a t u r e are considered. Urate, carbonate, oxalate, and phosphate s a l t s of the divalent cations are found as precipitates i n the lumen of the Malpighian tubules of many insects, including Ortho-pterans [reviewed by Clark, 1958]. Ramsay [1956], using an in v i t r o preparation of s t i c k insect (Orthopteran) Malpighian tubules, found ++ ++ that both Mg and Ca are present at much lower concentrations in Malpighian tubule f l u i d than in the hemolymph. In t h i s study, i t was found that the in v i t r o locust rectum has a very low permeability to C a + + from the apical side, but C a + + can enter the rectal tissue from the basal side much more readily (Table 2). The l a t t e r observation may suggest that Ca i s exchanged between the storage s i t e s within tissues and the hemolymph, thus maintaining a dynamic balance of free C a + + within the hemolymph of the locust. The amount of unbound C a + + in the hemolymph is ultimately regulated by the Malpighian tubules that secrete C a + + at ++ low rates. Ca may be continuously absorbed (actually concentrated ++ as i s Mg ; Wyatt, 1961) from digested food material in such large 3 9 quantities as to insure a steady supply of th i s divalent cation. In ++ e f f e c t , excess of Ca may be the normal s i t u a t i o n , and deficiencies exceptionally rare. -++ ++ The apparent lack of Mg uptake (at low Mg concentrations) by in v i t r o locust recta may r e f l e c t the in vivo s i t u a t i o n , since regulation (as suggested for Ca + +) perhaps occurs at the tissue and Malpighian tubule l e v e l s . At the hemolymph level a dynamic balance between bound (a f a i r l y large amount; Wyatt, 1961) and physiologically active Mg + + could control the amount of free Mg + + present throughout quite a wide range of ++ total Mg fluctu a t i o n . The Malpighian tubules excrete only a small amount of the total Mg + + present in the hemolymph [Ramsay, 1956]. This observation coupled with the observation that almost a l l insects, concen-++ trate Mg from t h e i r food [Wyatt, 1961] may not necessitate active retention of Mg + + by the rectum. PO^ does seem to be recycled because i t i s resorbed by the in v i t r o locust rectum. PO^ is accumulated as Pi in the basal compartment, and the rate of uptake i s affected by the phosphate concentrations in the apical and basal bathing media. This uptake i s against both e l e c t r i c a l and chemical gradients, cannot be explained by solvent drag and i s p a r t i a l l y inhibited by the presence of KCN and IAA. Harrison and Harrison [1961] studied the a b i l i t y of rat small intestine (in v i t r o ) to concentrate phosphate in the basal bathing medium. They found that t h e i r preparations were capable of producing B/A phosphate ratios of 4.2 - 5.4 to 1 af t e r 3 hours of incubation. They termed the uptake "true transport" of PO^, but did not characterize the transport mechanism further. An important consideration in the amount of Pi appearing in the basal compartment is the amount contributed by the tissue. The rectal 40 tissue i s capable of contributing a substantial amount of PO^ to the basal compartment, in the absence of PO^ in the bathing medium. This contribution by the rectal tissue decreases as the i n i t i a l concentration of PO^ in the basal bathing medium increases. P h i l l i p s [1964b, c] found + + that the in vivo locust rectum decreased i t s rate of uptake of Na , K , and C l ~ when the hemolymph concentration of these ions was increased. The observations on PO^ uptake in this study may r e f l e c t a s i m i l a r mechanism for control of phosphate uptake by the locust 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 rectal tissue to the basal compartment. As the concentration of phosphate in the basal bathing medium i s increased, the magnitude of the diffusion gradient from the tissue to the basal compartment i s decreased, and this 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 offers a plausible explanation of how PO^ moves from the tissues to the basal compartment, diffusion does not explain why poisoned recta, with large amounts of Pi in the tissues, contain less PO^ in the basal compartment than unpoisoned recta with small amounts of Pi in the tissues (Table 4) or why accumula-tion in the basal compartment i s dependent upon the PO^ concentration i n the apical bathing medium when the i n i t i a l levels of PO^ in the basal compartment are constant (Figure 10). Harrison and Harrison [1961] also noticed a tissue contribution to P0^ accumulation in the basal compartment of rat small i n t e s t i n e , but did not indicate how much, or how, phosphate moved from the tissue to the basal compartment. It was of interest to determine whether some of the phosphate that was found in the basal compartment was organic or not, because 41 hemolymph of some insects contains 20 - 30 mM/1 of acid-soluble organic-P0 A [Wyatt, 1961]. The rectal tissues perhaps contribute organic phosphates to the hemolymph; however, a l l measurable phosphates which were observed to be accumulated in the present experiments appeared as inorganic P0 A. 32 The tracer experiments indicate that P i s incorporated op into organic forms by rectal tissues and is.also transferred as Pi to the basal compartment. The d i l u t i o n of tracer P0 A by a large pool of unlabelled P0 A in the tissue means that tracer studies of t r a n s e p i t h e l i a l transport of P0 A are d i f f i c u l t to interpret. The effects of KCN and IAA, arsenate, and water uptake on accumulation of P0 A by rectal tissue and within the basal compartment, support the idea of a c a r r i e r on the apical border of the rectum, but do not exclude a c a r r i e r on the basal border as we l l . KCN and IAA i n h i b i t metabolic incorporation of P0 A in the rectal tissues, but do not seem to i n h i b i t the tissue uptake of Pi from the apical bathing medium (Table 3 and 4). Arsenate, a competitive i n h i b i t o r for P0 A uptake systems, decreases the tissue uptake of P0 A from the apical bathing medium. Together, the above two observations seem to indicate that a passive c a r r i e r system for P0 A uptake into the tissue exists on the apical border of the rectum. I f P0 A uptake in the basal compartment i s measured in the presence of water i n f l u x , the amount of P0 A found in the basal com-partment does not increase when the P0 A concentration in the apical bathing medium i s increased from 18 to 61 mM/1 of P0 A (top curve of Figure 10). This observation indicates that the saturable c a r r i e r i s the rate l i m i t i n g step for P0 A uptake in the rectum; however, where 42 this c a r r i e r i s located on the rectum i s not obvious. From a theoretical point of view i t would seem advantageous for the locust 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 apical 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 . Balshin [1973] found an active c a r r i e r for amino acids on the apical border of the locust rectum. PO^ ca r r i e r s have also been postulated for both the apical and basal border of an Orthopteran's Malpighian tubules [Maddrell, 1971]. 32 The large quantity of Pi incorporated into organic phosphates by rectal tissue may indicate that phosphorylation i s a key step in PO^ uptake, by'lowering the a c t i v i t y of Pi in the tissue. Goodman and Rothstein [1957] found that glyceraldehyde-dehydrogenase may be important in e s t e r i f y i n g PO^ as an aid to i t s entry into yeast c e l l s . Organelles, such as mitochondria, which are heavily concentrated at the apical border of the rectum, perhaps also aid in lowering Pi a c t i v i t y i n this part of the c e l l . Metabolic a c t i v i t y i s important in determining the amount of PO^ entering the basal compartment, because recta poisoned with KCN and IAA showed a marked decrease in PO^ accumulation in the basal compartment (Table 4). However, KCN and IAA do not greatly affect the tota l amount of PO^ entering the rectal tissue from the apical bathing medium (Table 3). It i s possible that PO^ uptake by the rectum i s a two step process, consisting of an entry step from the apical 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 translocation step across the c e l l into the basal compartment, mediated by metabolic incorporation 43 When a l l the observations are considered i t i s possible to put together a tentative model for PO^ uptake by the in v i t r o locust rectum. Although this model i s consistent with the findings of this study, i t is by no means the only explanation for the observations, but i s of value in so far as the experiments i t suggests. Apical Bathing Intima Rectal Tissue Basal Bathing Medium Medium Salient 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 apical border. This c a r r i e r i s inhibited by arsenate. Entry i s increased by high P0 4 concentration in the rectal lumen caused by more rapid water reabsorption [Speight, 1967]. (b) metabolic incorporation of PO^ in the rectal tissue. This step i s inhibited by KCN and IAA. 44 (c) PO^ transfer from the rectal tissue into the basal compart-ment i s mediated by either 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 either mechanism (ca r r i e r or diffusion) would be influenced by metabolic poisons because the l a t t e r cause large changes in tissue levels of inorganic PO^. The f a c i l i t a t e d entry step on the apical border of a c e l l , followed by metabolic incorporation 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. Scarborough [1970], studying glucose uptake in. Neurospora crassa, found a f a c i l i t a t e d d i f f u s i o n entry step for glucose. Glucose, upon entry into the c e l l s , was phosphorylated and then shunted into the general metabolism of the c e l l . When a non-metabolizable analog (3-0-methyl-D-glucose) was used as the substrate for the c a r r i e r , the concentration i n the c e l l s equilibrated with the bathing medium, but did not exceed that of the bathing medium (cell/medium glucose concentration r a t i o =1). Jain [1972] terms this type of uptake "loosely coupled energized transport," and although i t may not be an accurate description of P0^ transport in the locust rectum, the process seems to be analogous. Because of the presence of hormones and other factors jm vivo [Mordue, 1969], in v i t r o systems do not necessarily accurately r e f l e c t in vivo mechanisms, but i t i s s t i l l of value to extrapolate in v i t r o findings to the whole animal. Values are available for calculating the rate of P0^ secretion by the Malpighian tubles of the locust. Speight [1967] found that 45 primary Malpighian f l u i d contains 14.6 ± 5.65 nM/ul (mean ±S.D.) of PO4 (in vivo measurement). Maddrell and Klunsuwan [1973] found that an in v i t r o preparation of locust Malpighian tubules produced a primary f l u i d containing 12 nM/ul of P0 A. P h i l l i p s [1964c] found that the -Malpighian tubules of the locust secreted at a rate of 8 ul/hour. The estimated Malpighian tubule secretion rates for P0 A are 8 x 12 = 96 nM of P0 A/hour, and 8 x 14.6 = 116.8 nM of P0 A/hour. I f the rate of P0 A uptake into the basal compartment by recta incubated in apical bathing medium containing 18 mM/1 of P0 A i s taken from Figure 10, uptake values of 99.3 ± 7.65 nM of P0 A/hour/rectum (water flow across the rectal wall prevented), and 80.4 ± 11.2 nM of P0 A/hour/rectum (water flow across the rectal wall not inhibited)(values are mean ±S.E.) are obtained. The values for the Malpighian tubule excretion rates and rectal reabsorption rates for P0 A correspond quite closely. A l l of the P0 A excreted by the Malpighian tubules can be resorbed by the rectum. The locust rectum, therefore, has the resorptive c a p a b i l i t y expected of a s i t e responsible for P0 A regulation i n the locust. P0 A i s rapidly accumulated when hemolymph levels are low, and i s more slowly accumulated when hemolymph levels are higher. The saturation mechanism for P0 A uptake would allow excess P0 A to be voided with the feces should internal levels become too high. 46 SUMMARY 1. Net transfer of calcium or magnesium across the in v i t r o locust rectum was not observed. 2. Phosphate i s accumulated i n the basal compartment of the i n v i t r o locust rectum against large concentration difference. 3. The rectal tissue i s capable of contributing a substantial amount of inorganic P0 A to the basal compartment i n the absence of P0 A in the basal bathing medium. 4. Only inorganic phosphate (no organic phosphates) i s accumulated in the basal compartment. 5. Water uptake (solvent drag) does not increase the amount of P0 A accumulated i n the basal compartment. 6. The P0 A uptake f i t s Michaelis-Menton kinetics with of 5.0 mM/1 and V m a x of 52.6 nM/hr/mg D.W. rectal tissue. 7. P0 A entry into the basal compartment i s inhibited by 2 mM KCN/1 and 2 mM IAA/1. Arsenate i n h i b i t s P0 A entry from the apical bathing medium into the rectal tissue. 8. A possible mechanism for P0 A uptake by the rectum i s proposed. 47 BIBLIOGRAPHY BALSHIN, M. and J.E. PHILLIPS, 1971. Active absorption of amino acids in the rectum of the desert locust {Schistocerca gregaria). Nature New Biology. 233: 53-55. BALSHIN, M. 1973. Absorption of amino acids in the rectum of the desert locust {Schistocerca gregaria). Ph.D. Thesis, University 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 content in the hemolymph of certain insects. Physiol. 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 for the determination of tracer phosphate in biological material. A Chem. Scand. 4: 942-947. 48 GOH, S.L. 1971. Mechanism of water and s a l t absorption i n the in v i t r o locust rectum. M. Sc. Thesis, University of B r i t i s h Columbia, Canada. GOMORI, M.D. 1942. A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J . Lab. C l i n . Med. 27: 955-960. GOODMAN, J. and A. ROTHSTEIN. 1957. The active transport of phosphate into yeast c e l l s . J . Gen. Physiol. 40: 915-923. HARRISON, H.E. and H.C. HARRISON. 1961. Intestinal transport of phosphate: action of vitamin D, calcium, and potassium. Am. J. Physiol. 190: 1007-1012. JAIN, M.A. 1972. The bimolecular lipid membrane. Van Nostrand Rheinhold Company, New York. LEVINSON, C. 1971. Phosphate transport in Ehrlich Ascites tumor c e l l s and the effect of arsenate. J . C e l l . Physiol. 79: 73-78. MADDRELL, S.H.P. 1971. The mechanism of insect excretory systems, in "Advances in Insect Physiology." Ed. by J.W.L. Beament, J.E. Treherne and V.B. Wigglesworth, 8: 200-324. Academic Press, London, New York. 4 9 MADDRELL, S.H.P. and S. KLUNSUWAN. 1973. Fluid secretion.by in v i t r o preparations of the Malpighian tubules of the desert locust Schistocerca gregaria. J. Insect Physiol. 19: 1369-1376. MARTIN, J.B. and D.M. DOTY. 1949. Determination of inorganic phosphate Anal. Chem. 21: 965-967. MORDUE, W. 1969. Hormonal control of Malpighian tubule and rectal function i n the desert locust, Schistocerca gregaria. J. Insect Physiol. 15: 273-285. PHILLIPS, J.E. 1964a. Rectal absorption i n the desert locust, Schistocerca gregaria. Forskal, I. Water. .J. Exp. B i o l . 41: 15-38. PHILLIPS, J.E. 1964b. Rectal absorption in the desert locust, Schistocerca gregaria. Forskal, I I , Sodium, Potassium and Chloride. J . Exp. B i o l . 41: 39-67. PHILLIPS, J.E. 1964c. Rectal absorption in the desert locust, Schistocerca gregaria. Forskal, I I I . The nature of the excretory process. J. Exp. B i o l . 41: 69-80. PHILLIPS, J.E. 1965. Retal absorption and renal function in insects. 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 sieving of hydrophilic molecules by the rectal intima of 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 st i c k insect, Dixippus morosus, (Orthoptera, Phasimidae): calcium, magnesium, chloride, 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. Physiol. 46: 1075-1085. SCARBOROUGH, G.A. 1970. Sugar transport in Neurospora orassa. J. Biol Chem. 245: 1694-1698. SPEIGHT, D.I. 1967. A c i d i f i c a t i o n of rectal f l u i d in the locust Schistooeroa gregaria. M. Sc. Thesis, University 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 insect hemolymph. Ann. Rev. Ent. 6: 75-102. 

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