Some major transport mechanisms of insect absorptive epithelia

Camp. Biochem. Physiol. Vol. 90A, No. 4,pp.643-650,1988

0300-9629/88 $3.00f0.00

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SOME MAJOR TRANSPORT INSECT ABSORPTIVE

MECHANISMS EPITHELIA

OF

J. E. PHILLIPS,* N. AUDSLEY, R. LECHLEITNER,B. THOMSON,J. MEREDITH and M. CHAMBERLIN Department of Zoology, University of British Colombia, Vancouver, British Columbia, V6T 2A9, Canada (Received December

1987)

Abstract-l. After hormonal stimulation, fluid reabsorption

(J,) in locust hindgut from the KCI-rich, low-Na primary urine is driven primarily by an unusual mucosai electrogenic fi- pump (Jc,). 2. Cvclic-AMP increases J,-, and also mucosal K+ and basolateral Cl- conductances. so that KC1 absorpiion exceeds that of Na+ in rectum but not ileum. -1

3. Mucosal entry of Na+ occurs by exchange for NH: (H+), by cotransport with some neutral amino acids, and through putative channels. 4. However, transport of proline, the predominant organic substrate, is largely Na-independent and drives a sizable component of J, in rectal but not ileal segments. 5. There is evidence for hormonal control of Na+ reabsorption in ileum but not rectum.

INTRODUcTION Insects have a basic body plan consisting essentially of one simple epithelial tube, i.e. the gut and its outpushings (e.g. Malpighian tubules and salivary glands), within another, i.e. the cuticle-forming epidermis. Typically, these epithelia are unusual compared to those of most other animals in that they actively secrete and reabsorb KCl-rich fluids of lower Na+ content (reviewed by Phillips, 1981; Phillips et al., 1986) under the control of neuropeptide hormones (reviewed by Phillips, 1983). Insects live in as wide a range of extreme and fluctuating environments as do any other group of animals and they generally regulate their blood composition almost as well as vertebrates. The excretory process, which contributes to this homeostasis, is also unusual in that regulation is ultimately achieved by selective reabsorption primarily in the hindgut (ileum, colon, rectum) of many insects (e.g. locusts) from an isosmotic KCl-rich primary urine actively secreted by the Malpighian tubules. The percentage of primary urine reabsorbed in the hindgut varies between 0 and 100% in different species or under different conditions. In the desert locust, the final excreta may contain Na+, K+ and Cl- at very low (l-10 mM; salt-depleted individuals) or at very high osmotic concentration of levels (200-600 mM; 1000 mOsm; dehydrated, salt-loaded individuals) compared to the hemolymph (110, 10, 90mM respectively; 400 mOsm). The papillate recta of locusts, blowflies and cockroaches produce strongly hyperosmotic urine by a different mechanism from that in mammalian and avian kidneys. This involves absorption of a hyposmotic fluid, apparently as the result of solute-driven local osmosis and subsequent recovery of solute (i.e. ion recycling) within the rectal pad *To whom

all correspondence should work was funded by the Natural gineering Council of Canada.

by Phillips, 1977, 1981; Phillips et al., 1986; Gupta et al., 1980). We have concentrated on the rectum, and more recently the ileum of the desert locust (Schistocerca gregaria) to identify and characterize major membrane transport mechanisms in the apical and basolateral membranes of a typical insect absorptive epithelium and their hormonal control through second messenger systems (reviewed by Phillips et al., 1986). We anticipate that the epithelial model which we have developed for this preparation (Fig. 1) will serve as a prototype for other KCl-absorptive epithelia of insects, as the frog-skin has done for vertebrate epithelia. Earlier reviews have concentrated on mechanisms of Cl- and K+ absorption in locust rectum; therefore only the main characteristics of these processes are summarized herein. In this review we emphasize our recent work (in press or in preparation) on Na+ and proline absorption, NH,+ secretion and transport of acid-base equivalents in the rectum (i.e. lower half of Fig. 1), and on comparisons with mechanisms in the locust ileum. These topics are interrelated, because of the low Na+ levels in the hindgut which are available to drive the Na-coupled cotransport and countertransport mechanisms so common in vertebrate epithelia. How is this limited luminal Na+ used in apical entry processes? In particular, how is the main respiratory substrate for this epithelium, luminal proline, derived from Malpighian tubules (Fig. 2) transported and its oxidative end-product, ammonia, removed from this tissue? The locust hindgut may play a role in insect acid-base regulation analogous to that of the vertebrate renal tubule, i.e. eliminating excess H+ and reabsorbing HCO,, as required to maintain hemolymph pH nearly constant through the pCO,/HCO; buffer system in cooperation with the respiratory (tracheal) system (Phillips et al., 1986). Does ammonia trapping facilitate H + elimination and HCO; retention as it does in vertebrate kidneys and are these processes Na-coupled? Is Na+ re-

(reviewed

be addressed. This Sciences and En-

643

644

J. E. CELL

LUMEN

Ion Activltier Na*

HEMOCOEL

(mM) and PD (mV)

g

K*

0

75

70

7.2

cl-

52

47

82

Proline

13

06

13

VN

I

7.35

7

mV

l54

0

+34

Net Electrochemical

PD

(mV)

(Bmolateral)

(Apical)

Na’

PHILLIPS

127 (Iavoring

)

127 (ocqoain~)

K*

12 (favoring)

20

(favoring

cl-

50

(oPPo8lW)

20

(favorinp)

n+

55

(OPDmlW)

56

(lavoring

) )

Fig. 1. Model showing mechanisms identified in apical and basolateral membranes of locust rectal pad epithelium (basolateral exit mechanisms for amino acids, AA and acid-base equivalents unknown). The neuropeptide hormone, CTSH, acts via CAMP to stimulate @ or inhibit @ four mechanisms: thick arrows, major ion pumps; thin arrows through solid circles, carrier-mediated co- or counter-transport; arrows through gaps, ion channels. No more than 20% of AA absorption and H+ secretion are Na-coupled. Steady-state values given for net transepithelial flux. (JT;;) and electrochemical potential differences across the two cell borders (favouring or opposing net fluxes) are for stimulated recta in Ussing chambers and bathed bilaterally in control saline under open-circuit conditions, except short-circuited state for Na + and AA, and unstimulated state for NH: and Na+ (CAMP has no effect), H+ and AA (CAMP inhibits net fluxes of H+ and proline, but not glycine). After stimulation, estimates of relative permeabilities (P x lO’cm/s) are P,sP,, > PC, (17: 1:0.5) for apical and P, = PC, > P,, (5 : 5 : 0.5) for basolateral borders, Modified and updated from Phillips et (11. (1986).

absorption in insects under neural or hormonal control, as previously shown for KC1 and fluid? In this paper we review our recent progress in addressing these major questions. METHODS

The methods used to study fluid and solute absorption and their control across locust rectum in situ and in ritro have been reviewed in detail by Hanrahan et ul. (1984) and for acid-base and ammonia transfer by Thomson c/ al.

el a/.

(1988). More recently we have successfully applied similar methods during studies of locust ileum. Both these gut segments have been mounted as fat sheets between modified Ussing chambers (oriface 0.2 cm’) under open or shortcircuited conditions to measure transepithelial short-circuit current (I,), potential (V,) and resistance (R,) values which usually remain stable (steady-state) for > 8 hr after an initial transient phase (2 hr). Unidirectional fluxes of radioisotopes (.I;;, to hemocoel side; Jr;, to lumen side; 5:61, calculated by difference) were measured at 20 min intervals for a few hr to obtain steady-state estimates of active and passive solute movements. Cable analysis (Hanrahan and Phillips. 1984~) has shown that rectal epithelium is a tight epithelia with >90% of ion flux by the transcellular route after stimulation with I mM CAMP added to the serosal side. Similar gross electrical properties suggest that the situation may be the same for the locust ileum. These hindgut preparations also provide good bioassays to identify and purify putative hormones controlling absorption in the hindgut. To localize and characterize specific mechanisms within rectal epithelium, the electrochemical potentials favouring or opposing net flux of specific ions across the apical (Apt,,) and basolateral (Aph,) plasma membranes were measured by impaling epithelial cells with double-barrelled ionselective glass micro-electrodes from either the lumen (e.g. rectum with cuticle cut) or hemocoel (e.g. ileum) sides. Deflections (A) in apical (V,) and basolateral (V,) membrane electropotentials caused by transepithehahy applied constant-current pulses were used together with ion substitutions in the external saline to estimate ion conductances of each membrane (G,(, G, respectively). To study net movement of acid base equivalents (J,“,,, Jzty) to the lumen or hemocoel side (i.e. not necessarily transcellular), Ussing chambers (2 ml volume) were perfused bilaterally (5 ml/min) for 2 hr to ensure a steady-state, The perfusion was stopped on one side only and JzJ: or ./I,‘,, was continuously monitored using a ‘Radiometer pH-stat’ as the amount of HNO, or NaOH required to maintain external pH at a pre-selected value. Net ammonia movement to the lumen side (J;:“) was determined using a similar protocol and measuring the increase in external concentration (2@200 PM range) spectrophometrically. as described by Kun and Kearney (1974). Although fluid entering locust hindgut in situ normally contains (mM) 140 K + . 90 IIOCI-, 2@46Na’ and 38 proline as the main solutes, recta were bathed bilaterally under our normal (control) conditions in a complete saline resembhng locust hemolymph (including I IO Na’ , IO K +, 100 Cl-, 12 proline, pH 7) and containing I I amino acids and glucose at hemolymph levels (Hanrahan er ut.. 1984). This was necessary to satisfy short-circuit conditions of no concentration difference across the epithelium. Cl was replaced by methylsulphate or gluconate, Na+ by choline or N-methyl-D-glucamine, and K + by Nat, in various ionsubstituted salines. Salines normally contained IO mM HCO< and were stirred by 95% 0,: 5% CO? gas. Bicarbonate was replaced by MOPS buffer and 100% O2 gas was used in HCO, /COz-free salines. All experiments were conducted at 22 + 1 C unless stated otherwise. Rates of fluid transport (J,) and absorbate composition were monitored hourly over 5 4 hr by weighing cannulated everted sacs (rectum or ileum) and by completely removing the fluid inside sacs for analysis of its composition as previously described (Goh and Phillips. 1978). Ion absorption rates (_I’““. ne,, i.e. open-circuit) were calculated from J, and absorbate concentrations. Sacs absorb from normal saline (lumen side) equally well whether they are filled hourly on the hemocoel side with fresh normal saline (I-10 ELI,i_ stimulants) or not (i.e. allowed to bathe serosally in their own absorbate). JFz was determined with only sufficient saline (I ~1) placed initially inside sacs to supply the stimulant.

645

Transport of insect absorptive epithelia \

active riecretionof proline into Malpighian tubules

glycolysis

/ /

\ LUMEN

\

proline

Fig. 2. The system whereby rectal metabolism and I, are sustained by the main respiratory substrate, fuminal proline (concentrations measured in body compartments shown), supplied from Malpighian tubules (upper). Local events at apical membrane infoldings where proline enters actively to fuel mitochondria, thereby providing ATP for the apical Cl- pump (I,) and hence fluid transport. while ammonia from proline oxidation is secreted into the lumen (lower left). The metabolic pathway for rectum elucidated by enzyme analyses, substrate utilization and inhibitor studies (lower right). Based on data in Chamberlin and Phillips (1982a,b; 1983) and Thomson et al. (1988).

RESULTS

Trans~pithe~iai electrical properties ~nd~uxe~: actions of ~timuIants After removing recta or ilea from locusts to control saline, the large I,, and V, indicating net anion transport to the hemocoel (serosal) side (as also observed in situ) fall close to zero (I, of + 1 peq,i,/cm2/hr; L’, of - 1 mV for ileum, 7 mV for rectum). Serosal addition of storage or glandular lobes of corpus cardiacum (CC) containing the neuropeptide CTSH, or l-5 mM CAMP, or 50-100 GM forskolin to such recta and ilea rapidly increases IX back to about 10 K,+,~/cm*/hr, and Y( to 32 (rectum) and 21 (ileum) mV. These high Z, and k’[values then persist for many hours. Concurrently, R, decreases after stimuIation from 280 to 160 (rectum) and 240 to 98 (ileum) Rcm2. The ileum, unlike the rectum, is equally stimulated by a peptide in homogenates of ventral abdominal ganglia (3rd to 7th), which differs from CTSH of CC in that Z,, declines to baseline again with 2 hr. Stimulant-induced A& is completely accounted for by increases in net active flux of 36Clacross recta to the hemocoel side and the A[, across both gut segments is completely abolished in Cl-free salines. These observations indicate that the neuropeptide stimulants in locust CC and VG act on adenylate cyclase to increase intracellular CAMP, which then stimulates electrogenic Cl- transport (reviewed by Phillips et at., 1986 for rectum; ileum, unpublished observations). In support (Table l), Chamberlin and Phillips (1988) and Herault and

Proux (1987) both recently observed a transient increase in rectal CAMP levels, peaking about 10 min after adding CC when the rate of 1, increase is most rapid. Forskohn doubled rectal tissue CAMP levels, whereas theophyliine caused only small increases, consistent with the lesser effect of this agent on Z, (Table 1). However, theophylline does prolong the elevation of tissue CAMP caused by CC. We have obtained no evidence that Ca2+ is involved in this response system but tissue cGMP does increase slowly over the first hr after adding CC, and may be involved in sustaining I,, after CAMP levels have returned to normal (by 30min). Under short-circuit conditions, CAMP causes a 4-fold increase in both unidirectional fluxes of %+ across locust rectum, indicating a large increase in Table 1. Effect of stimulants on rectal short-circuit current (I,) and tissue CAMP content after IS min Stimulant (dosage) Control

Increase in I, (&,,,,lcm’ihr) --

Tissue CAMP (pmol/mg wet/W)

0

0.29

CTSH (0.1CC/ml) CTSH (0.I CC/ml)

6.6* 7.1’

0.46’ (0.8I)? 0.431

~-

+Theophylline (4 mM) Theophylline (4 mM) Forskolin (50 PM) Recent dissection of recta (fed or starved)

2.7’ 6.2+ 6*

0.33 0.66* 0.84;

*Signific~ntiy different from control values. fPeak value at 10min. Standard errors were 10 to 20% of means (n = 4-6: Cham~rlin Phillips. 1988): CC = corpus cardiacurn gland equivalent.

and

3. E. PHILLIPS et al.

646

passive permeability to this cation but with only a small active net flux which is less than 10% of Jzi,. However, under open-circuit conditions when the Cl- pump is allowed to develop a large Y,, net flux OF42K+ equals that of 36C1- (Fig. I) due to electrical coupling. This passive net K+ flux exceeds the active net flux of “Na+ (Jr$) across recta by 2-fold even though the K+ :Na+ ratio in the saline is 1: 11 (Fig. 1). While CAMP and neural tissue extracts do not affect rectal JFl (short-circuited state; Spring and Phillips, 1980; Black ef al., 1987) these stimulants and also VG all stimulate net absorption of NaCl much more than KC1 across everted ileal sacs (Fig. 3) whether the luminal K+:Na+ ratio is 1O:llO or 110:10mM. Concurrently the anion deficit (Na+ + K+ - Cl-) in ileal absorbate, which is probably accounted for by HCI -, largely disappears after stimulation with CC or VG (Fig. 3). These recent observations on locust ileum provide the first direct evidence for neuroendocrine factors controlling Na + reabsorption in insect excretory systems (Lechleitner and Phillips, in preparation). In the absence of an initial osmotic concentration difference, unstimulated everted ileal and rectal sacs actively absorb fluid (J,) at a similar rate

Stimul8tion

(7-8 ~l/~rnzlhr), as a secondary consequence of ion transport (i.e. local osmosis; Fig. 3). In support of this view, stimulants of ileal and rectal NaCI and KC1 transport have a parallel effect on active fluid absorption, thereby increasing rectal J, to 16 and ileal J, to 16(+cAMP) and 29(+CC or VG) pl/cm’/hr (Fig. 3). Rectal AJ, caused by CAMP is abolished in Cl-free salines, as expected if antidiuretic (ADH) activity in CC were due to CTSH acting on Jzi,. The results in Fig. 3 suggest that ileal reabsorption is probably more important in the overall excretory process than previously supposed although macroscopic surface area of the rectum is 50% greater. Unlike unstimulated everted rectal sacs which absorb a fluid that is 25-30% hypo-osmotic to the luminal saline (Phillips et al., 1982; Phihips, 1977) ileal absorbate is consistently slightly hyperosmotic to control saline by 3.5% before and 10% after stimulation (Lechleitner, unpublished observation). This difference in osmoiarity correlates with the absence in ileum of elaborate lateral intercellular channels, which are believed to be the sites of ion recycling leading to hyposmotic rectal absorbate (reviewed by Phillips et al., 1986). With all luminal Na+, K+ and Cl- replaced by

of Absorption

(open

Everted

Rectum

circuit

f

lleal

(0.64cm2f

Sacs+tFlat

Sheet

Preparations

z 02 JV

JS’net

J*a net

JKnet

J”net

Fig. 3. The maximum mean effect of stimulation by CAMP (hatched bars) or by corpus cardiacum (CC) or ventral abdominal ganglia (VG) extracts (stippIed bars; CC and VG effects similar) on fluid (2,) and ion absorption (J$, Jtz, J,“,,) an d on acid secretion (Jn) compared to unstimutated control locust ilea or recta (open bars); identical control salines initially present bilaterally under open-circuit conditions, except short-circuit state (S -C) for rectal JfG. Unstimulated fluid transport from luminal solution containing 80 mM proline but no Na ’ , K+ and Cl- shown by salid bars. The mean changes in rates shown after stimulation are all highly significant, except rectal Jz$ (standard errors all less than 20% of mean values). Data for locust rectum from Hanrahan and Phillips (1983; K+, Cl-), Black et al. (1987; Na’). Thomson and Phillips (in preparation; H+), Proux et al. (1984; J,), and proline-driven J, (Lechleitner and Phillips, in preparation). Data for locust ileal sacs from Lechleitner and Phillips (in preparation), except for J7c, across preparations in Ussing chambers (Thomson and Phillips, in preparation).

Transport

of insect absorptive

osmotically equivalent sucrose, proline (4&80 mM) alone sustains J, at about 50% of control rates across rectal sacs (Fig. 3). In the presence of these ions, high luminal proline also increases 1, across rectal but not ileal sacs (Lechleitner, data not shown). Apical and basolateral membrane mechanisms for CI-, K + and Na + in locust rectum

Under all conditions (e.g. open-current, Fig. 1; short-circuited; Hanrahan and Phillips, 1982, 1983, 1984~) apical entry of Cl- into epithelial cells occurs against both large electrical and net electrochemical potentials, but down a small concentration gradient (2-fold). Clearly the mucosal entry step is active, whereas there is always a A& of about 20mV favouring passive exit of Cl- across the basolateral membrane (Fig. 1). Cyclic AMP causes the apical electrogenic Cl- pump to transport Cl- into the cell 10 times faster and against both a larger A@, , which increases by 18 mV, and a V,, which increases by 7 mV. We concluded therefore that CAMP apparently does not influence the Cl- pump indirectly (e.g. on cation entry) by lowering the opposing potential. Stimulation causes a large increase in Cll conductance across the basolateral border, consistent with opening of Cl- channels. In support, Cl- channel blockers added serosally inhibit Cl- dependent 1, (Phillips et al., 1986). In essence the basolateral membrane, which is K+ selective (i.e. containing Ba-sensitive K+ channels; Hanrahan et al., 1986) in the unstimulated state, becomes equally permeable to Cl- and K+ after CAMP is added. A third action of intracellular CAMP is to open Ba-insensitive K+ channels, which are inhibited by high luminal K+ and anoxia, in the apical membrane. There are small electrochemical gradients favouring passive K+ absorption across both cell borders and these both increase after stimulation but are near zero under short-circuit conditions (Fig. 1; reviewed by Phillips et aI., 1986). In the absence of external Cll and with a hemocoel-to-lumen K+ gradient (80: 10 mM with V, clamped at 0 mV), serosal addition of CAMP to recta induces a large K+ diffusion current to the lumen side, which is blocked by serosal but not mucosal addition of Ba2+ (Hanrahan et al., 1986). We have recently obtained nearly identical results with locust ileum (Audsley, in preparation). This suggests that the mechanisms and control of K+ absorption are similar in locust ileum and rectum (Fig. 1), although K+ absorption appears to be quantitatively less important in the former gut segment (Fig. 3). The apical electrogenic Cl- pump in the rectum is different from NaCl (or Na+, K+, 2Cll) cotransport and HCO; /Cll or Na+, HCO; /Cl- exchange mechanisms reported in other membranes (Hanrahan and Phillips, 1982, 1983, 1984a-c 1985). Rectal JFi, is stimulated by low luminal levels of K+ at an external site, but is not affected by complete absence luminally of Na+, HCO;/C02, M g2+, Ca2+ or by pH changes from 6 to 8. Increasing luminal K+ from 10 to 100 mM increases both K, (60 mM) and J,,,,, (15 ~,,,~,/cm~/hr) for Cll transport by about 5-fold under short-circuit conditions. KC1 cotransport has been excluded because the CAMP-induced increases in both unidirectional fluxes of 42K+ persist in Cl-free salines. The locust Cl- pump is highly selective, with

647

epithelia

only Br- partially substituting (50%) for Cl-. Hill plots indicate non-cooperativity between Cl- sites. manipulation of apical electroExperimental chemical gradients for Na+, K+ and HCO; indicate that these are not potential energy sources to drive apical Cl- transport in locust rectum (Hanrahan and Phillips, 1982, 1983, 1984a-c). The available data would also seem to exclude apical H+ and OHgradients (Phillips et af., 1986) but coupling of these forces to Cl- transport has not been fully explored. We have therefore been forced to consider that rectal Cl- transport may occur by an anion ATPase, which is still a controversial concept (see Gerencser in this volume). Lechleitner and Phillips (1988) have observed enrichment of an anion-stimulated ATPase in a microsomal fraction from locust rectum. This fraction was also enriched several-fold in plasma membrane markers and had low levels of mitochondrial enzymes. Despite this encouraging biochemical evidence for anion ATPase in plasma membranes of the rectum, the mechanism of energy coupling between rectal Cltransport and metabolism has not been established. The kinetics of stimulated Cl-dependent 1, in the ileum and the responses of 1, to luminal ion substitutions (i.e. Na+, HCO; /C02, K+ replacements) are similar to those reported for the rectum. This suggests that the same type of Cl- transport mechanism is present in both these hindgut segments (Irvine et al., in preparation). Lechleitner and Phillips (1988) also showed that Na, K-ATPase activity was concentrated in lateral membranes (as predicted in Fig. 1) which co-migrate with a mitochondria-rich fraction. Intracellular recordings indicate that the active step for transepithelial net transport of Na+ (K, of 17 mM; J,,,,, of 1.5 to 2.5 ,u(equiv/cm2/hr)rwhich is partially inhibited by ouabain and amiloride (1 mM) and which is Clindependent, is definitely at the basolateral border (Hanrahan and Phillips, 1984~; Black et al., 1987). Consequently, there is a large Ajik available to drive Na-coupled cotransport processes at the apical membrane (Fig. 1) even at physiological levels (e.g. Api, of 80 mV at 30mM luminal Nat; Hanrahan and Phillips, 1982, 1984c). Amino acid reabsorption, secretion

metabolism

and ammonia

As summarized in Fig. 2, locust Malpighian tubules actively secrete proline (38 mM). This constitutes 80% of total amino acids entering the hindgut where most of the proline is actively reabsorbed. Luminal proline is the predominant respiratory substrate supporting rectal Z, (Chamberlin and Phillips, 1982a,b). The metabolic pathway for proline oxidation in this tissue has been elucidated based on enzyme levels, substrate utilization by isolated mitochondria and by whole rectal tissue, and inhibitor studies (Chamberlin and Phillips, 1983). Both transamination and complete oxidation of proline occur but the latter pathway predominates in shortcircuited recta in oitro, resulting in substantial ammonia production. Thomson et al. (1988) have recently confirmed that amino acids absorbed from the lumen are the predominant source of ammonia production in locust rectum and 90% of this ammonia is prefer-

648

J. E.

PHILLIPS et al.

entially secreted into the lumen (0.6 pequlv/cm*/hr). The properties of the mechanisms responsible for amino acid transport and ammonia secretion back into the lumen (Fig. 2) are discussed below. Balshin and Phillips (1971; also Spring and Phillips, 1984; reviewed by Phillips et al., 1986) demonstrated transepitheiial active absorption of five neutral amino acids against 3 to IO-fold concentration differences when fluid absorption was prevented. Of these amino acids, proline and glycine were by far the most rapidly transported. Tissue levels (e.g. 60--70 mM proline) always exceeded external concentrations by at least 2-fold, indicating that the active step for amino acid transport is at the apical membrane. Uptake of alanine and serine were strongly stereospecific for L-isomers (proline not tested) and serine was a competitive inhibitor of glycine influx. Influx of glycine into rectal tissue obeyed Michaelis-Menten kinetics and was 80% dependent on the presence of luminal Na+, indicating the presence of a Na-glycine cotransport process. Na+ removal doubled I( from 22 to 43 mM without changing J,,, of “C-giycine influx. However, in the physiological range (e.g. IOmM) net flux of glycine across short-circuited recta, which is not changed by adding CAMP, is relativety low (0.13 fi_v/cmz/hr) and can only account for 5 to 10% of net Na+ influx. Proline is by far the major organic molecule reabsorbed in locust rectum as determined by fluxes across short-circuited preparations. This occurs, with only 10% of proline metabolized to other forms, by a remarkably high capacity ( k’,,,,Xof 4.2 p,suiv/cm2/hr) and high affinity (K, of 10mM) system and at high flux ratios (40: 1) compared to other transport processes in this epithelium. Only J:i, after stimulation exceeds active f$” at physiological concentrations (i,e. .Q$*” of 3.8~~~“~crnz/hr at 38mM luminal proline; Meredith and Phillips, 1988). The value for amino acid absorption given in Fig. 1 represents the decrease in ,i$in’ at 12 mM caused by prolonged (5 hr) bilateral absence of Na+. Replacement of only luminal Na+ recently indicated that at most 0.5 pequiv/cm2/hr of total Jiz”“” (2.1 p,,,,,/cm*/hr under control conditions) might possibly be Na-coupled. Given that J6”“” exceeds Jfz at natural concentrations by at least 2-fold, it is not surprising that proline transport is 80% Na-independent (not shown in Fig. 1). The evidence against Na-proiine cotransport is as follows: (a) absence of luminal Na+ (i.e. reversing Apia) only decreases Jgyline temporarily by 20% for 1 hr. (b) Changing praline influx ICfold by reducing luminal levels of this amino acid does not alter the influx of **Na+ (measured at K, value for Na+ transport of 17 mM). (c) As expected for cation--proline cotransport, rapid changes in luminal proline from 0 to 30mM reversibly depolarizes V, but not V, by 12 mV and also increases G, by 30 Rem*. However, these electrical changes occur whether luminal Na+ is present or not. The Naindependent inward current of cations associated with proline transport was calculated to be 1.5 pCqUIY/cm2/hr, or 43% of J$nc at 30 mM. Rapid decreases in luminal pH from 7 to 6, which are known to double the large A& favouring proton influx (see Fig. 1) does increase Jgline by 34%.

Therefore a proline-proton cotransporter may acbut other interpretations of count for part of J,,,proline these results have not been excluded. Complete replacement of luminal Cl- or KC does not change JP’“““; moreover, there is negligible A& to drive a G-proline cotransporter in locust rectum (Meredith and Philhps, 1988). Although both locust hindgut segments actively secrete acid under open-circuit conditions (Figs 1 and 3). ammonia secretion Jam, (cell to rectal lumen) apparently occurs largely by exchange of NH,’ for Na+ rather than by diffusion trapping of NH, in the acidic lumen. The evidence is as follows: (a) when Iuminal pH (HCO; /CO,-free saline) is reduced stepwise from 7 to 5 while keeping serosal pH at 7, acid secretion falls linearly from 1.5 p,qu,v/cm2/hr to zero, remains constant at 0.6 p,+/cm*/hr. This is yet Jamm true even when buffering capacity is greatly increased to exclude a pH disequilibrium in unstirred layers. (b) J*,, is inhibited 60% by either adding 1 mM amiloride or removing ail luminal Na+. (c) Changes in V, or Yt have no effect on J,,, as expected for a neutral cation exchanger. (d) Absence of luminal K+ or Cidoes not reduce luminal Ja,,,:,,,significantly (Thomson et al., 1988). Mechanisms of Na + entry in apical membrane

Na+ permeability of the apical membrane in locust rectum is very low compared to that of K+. For example, a 50-fold change in lumen Na+ level only changes V, in stimulated recta by 3 mV compared to 50 mV per decade change in K+ concentration (Hanrahan and Phillips, 1984~). The situation is not very different for unstimulated recta (Black et al, 1987). In the absence of any exogenous amino acids to sustain J,, or other luminal organic substrates to support Na-cotransport, V, is decreased reversibly by 6 mV when luminal Na+ is rapidly increased from 0 to 110 mM. Moreover, a reduction in voltage divider ratio (AY,/AV,) indicates an increase in apical G, (possibly via a Nat channel). Under control conditions, this substrate-independent conductive entry of Na+ (0.8 pesuiv/cm2/hr) accounts for 3&40% of JFi. Apical Na+ exchange for NH: (up to 0.6 p,,i,/cm2/hr) and for H+ (0.2 pLeqUiv/cm2/hr; see below) accounts for an equal fraction of J$. The remainder of Jz may occur by the small Nadependent fraction of JIEline (0.5 pcqurv/cm’/hr; Black et al., 1987) and by Jfp. Transport of acid-base e~u~l~~Ients

Surprisingly, hemolymph pH regulation in insects has been largely neglected by comparative physiologists. However, by analogy to vertebrates, it seems likely that this is achieved through regulation of hemolymph pCOZ and HCO, levels by the tracheal and excretory systems respectively (Phillips et al., 1986; Harrison, 1985). Luminal fluid becomes increasingly acid (falling from pH 7.9 to 5.0) as it passes through the hindgut and this is increased after imposing an acid load on the hemolymph. Protons are actively secreted across the apical membrane in the rectum against both electrical and concentration differences, for an opposing A,& of 86 to 100 mV (Fig. 1; Thomson and Phillips, 1985; Thomson et al., 1988). This open-circuit f$, (1.5 ~=~“i”~cm2~hrwhen

Transport

of insect absorptive

pH is 7.0) is electrogenic, inhibited by 1 mM azide, and stimulated 50% by including HC0,/C02 in the saline. J$ declines linearly to zero when luminal pH is reduced progressively to 5.2 (hemocoel side held at pH 7.0). Alkalinization occurs below this luminal pH. Inhibition by adding 1 mM amiloride or removing luminal Na+ both indicate that only 20% of Ce, may occur by Na+/H+ exchange. A 50% inhibition of Jr’, caused by replacing all luminal K+ can be explained by hyperpolorization of I’., by 2-fold. There is not sufficient energy in Api to drive proton-secretion by H+/K+ exchange. Jre, is not changed following complete replacement of external Cl-, or by adding the HCO, /Cl- exchange inhibitor, I mM SITS or the carbonic anhydrase inhibitor, 1 mM acetazolamide. The locust rectal proton pump has properties very similar to the pump described for turtle bladder and mammalian renal collection ducts. Acid secretion in locust rectum is accompanied by an equal movement of base equivalents (OH-, HCO,) to the hemocoel side by unknown mechanisms in the basolateral membrane. Using unstimulated rectal sacs bathed in control saline (i.e. HCO, /CO1 present), bicarbonate absorption (J:z”) was 0.4 p,qu,v/cm’/hr (Fig. l), which accounts for much of the anion deficit (Na+ + K+ -Cl-) in the absorbate, which is evident in Fig. 3. JLyol is completely inhibited by DIDS serosally and by acetazolamide, suggesting that basolateral exit of HCO; occurs in exchange for Cl--. Such a basolateral location for electroneutral HCO, /Cl- exchange has been conclusively demonstrated in posterior rectum of saline-water mosquito larvae (Strange and Phillips, 1985). Stimulation with CAMP under open-circuit conditions drastically reduces active secretion of protons by recta (by 66%) and also in ileum mounted in Ussing chambers (Fig. 3; Thomson and Phillips, in preparation). In agreement with this finding, stimulating locust ileal sacs with CC or VG extracts completely eliminates the anion deficit in the absorbate. (Fig. 3) as predicted if both J,“,, (secretion) and J,“,‘j (absorption) were inhibited; i.e. increased Cltransport compensates for decreased J$O3. We have still to examine how this apparent neuroendocrine control of acid-base transport across locust hindgut might be implicated in regulation of hemolymph pH. bilateral

REFERENCES

Balshin M. and Phillips .I. E. (1971) Active absorption

of amino acids in the rectum of the desert locust (&histocerca gregaria). Nature, Lond. 233, 53-55. Black K., Meredith J., Thomson B., Phillips J. and Dietz T. (1987) Mechanisms and properties of sodium transport in locust rectum. Can. J. 2001. 65, 30843092. Chamberlin M. and Phillips J. E. (1982a) Metabolic support of chloride-dependent short-circuit current across locust rectum. J. exp. Biol. 99, 349-361. Chamberlin M. E. and Phillips J. E. (1982b) Regulation of hemolymph amino acid levels and active secretion of proline by Malpighian tubules of locusts. Can. J. Zoo/. 60, 2745-2752. Chamberlin M. E. and Phillips J. E. (1983) Oxidative metabolism in the locust rectum. J. Camp. Physiol. B 151, 191-- 198. Chamberlin M. E. and Phillips J. E. (1988) EtTects of

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stimulants of electrogenic ion transport on cycle AMP and cyclic GMP levels in locust rectum. J. exp. Zool. 245, 9-16. Goh S. L. and Phillips J. E. (1978) Dependence of prolonged water absorption by in vitro locust rectum on ion transport. J. exp. Biol. 72, 25-41. Gupta B. L., Wall B. J., Oschman J. L. and Hall T. A. (1980) Direct microprobe evidence of local concentration gradients and recycling of electrolytes during fluid absorption in the rectal papillae of Calliphora. J. exp. Biol. 88, 2148. Hanrahan J. W. and Phillips J. E. (1982) Electrogenic K+-dependent chloride transport in locust hindgut. Phil. Trans.-R. Sot. Lond. B 299,-585-595. Hanrahan J. W. and Phillins J. E. (1983) Mechanism and control of salt absorption in locust’ rectum. Am. J. Physiol. 224, R131-R142. Hanrahan J. W. and Phillips J. E. (1984a) KC1 transport across an insect epithelium. Characterization of Kstimulated Cl absorption and active K transport. J. exp. Biol. 111, 201-223. Hanrahan J. W. and Phillips J. E. (1984b) KCI transport across an insect epithelium. I. Tracer fluxes and the effects of ion substitution. J. Membrane Biol. 80, 15-26. Hanrahan J. W. and Phillips J. E. (1984~) KC1 transport across an insect epithelium. Il. Electrical potentials and electrophysiology. J. Membrane Biol. 80, 2747. Hanrahan J. W. and Phillips J. E. (1985) Further observations on the regulation of KC1 absorption across locust rectum. J. exp. Biol. 116, 153-167. Hanrahan J. W., Meredith J., Phillips J. E. and Brandys D. (1984) Methods for the study of transport and control in insect hindgut. In Measurement of Ion Transport and Metabolic Rate in Insects (Edited by Bradley T. J. and Miller T. A.), pp. 19-67. Springer, New York. Hanrahan J. W., Wills N. K., Phillips J. E. and Lewis S. A. (1986) Basolateral K channels in an insect epithelium: channel density, conductance, and block by barium. J. Gen. Physiol. 87, 443466.

Harrison J. M. (1985) The effect of temperature on hemolymph pH in the american locust. Am. Zool. 25, 118A. Hera& J. P. H. and Proux J. P. (1987) Cyclic AMP: The second messenger of an antidiuretic hormone from the glandular lobes of the migratory locust corpus cardiaca. J. Insect Ph ysiol. 33, 487-49 1. Kun E. and Kkarney E. B. (1974) Ammonia. In Methods of Enzvmic Analvsis. Vol. 1 (Edited bv H. U. Beremever). pp.‘180221806. Academic Press, New York. _ ” Lechleitner R. and Phillips J. E. (1988) Anion-stimulated ATPase in locust rectal epithelium. Can. J. Zool. (in press). Meredith J. and Phillips J. E. (1988) Sodium-independent proline transport in the locust rectum. J. exp. Biol. (in press). Phillips J. E. (1977) Excretion in insects: function of gut and rectum in concentrating and diluting the urine. Federation Proc. 36, 248G2486. Phillips J. E. (198 1) Comparative physiology of insect renal function. Am. J. Phvsiol. 2.41, R241lR257. Phillips J. E. (1983) Endocrine’control of salt and water balance: excretion. In Endocrino1og.v of Insects(Edited by Laufer H. and Downer R.I. no. 411425. Alan R. Liss. New York. Phillips J. E., Hanrahan J., Chamberlin M. and Thomson B. (1986) Mechanisms and control of reabsorption in insect hindgut. Adtl. Insecl Physiol. 19, 329422. Phillips J. E., Meredith J., Spring J. and Chamberlin M. (1982) Control of ion reabsorption in locust rectum: implications for fluid transport. J. exp. Zoo/. 222, 297-308. Proux B., Proux J. and Phillips J. (1984) Antidiuretic action of corpus cardiacum (CTSH) or long-term fluid absorption across locust recta in vitro. J. exp. Biol. 113,40942 I. I.

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PHILLIPS et al.

Spring J. H. and Phillips J. E. (1980) Studies on locust rectum-II. Identification of specific ion transport processes regulated by corpus cardiacum and cyclic-AMP. J. exp. Biol. 86, 225-236.

Spring-J. and Phillips J. E. (1984) Proline transport and oxidation in short-circuited locust rectum: effect of CAMP. Can. J. Zool. 62, 1732-1736. Strange K. and Phillips J. E. (1985) Cellular mechanism of

HCO; and Cl- transport in insect salt gland. J. Membrane Biol. 83, 25-31.

Thomson B. and Phillips J. E. (1985) Characterization of acid/base transport in an insect epithelium. Fed. Proc. 44, 643. Thomson R. B.. Thomson J. M. and Phillius J. E. (1988). NH: transport in an acid secreting epithelium. km. j. Physiol. 254, R348-R356.