ABSTRACT
Long-term fluid absorption (Jv) by everted rectal sacs from locusts is stimulated by both corpus cardiacum (CC) extracts and cAMP in a dosedependent manner. This hormonal antidiuretic activity (ADH) is present in both nervous and glandular lobes of CC. This distribution is similar to that of chloride transport stimulating hormone (CTSH) but not to that of other factors previously reported from this gland. As expected if ADH were due to CTSH acting on electrogenic Cl- transport, CC extracts also increased the electropotential across rectal sacs, and the stimulation of fluid absorption ceased in Cl-free salines. CC extracts also caused recta to absorb fluid against larger osmotic gradients, suggesting that the antidiuretic factor acts on the ion-dependent active transport of fluid rather than on the osmotic permeability of the rectal wall.
INTRODUCTION
Regulation of Malpighian tubule excretion by diuretic hormones in insects is now well known. There are also several preliminary reports of putative diuretic and antidiuretic factors influencing fluid reabsorption by isolated insect recta (reviewed by Phillips, 1981, 1983) but the validity of the in vitro methods used in these early studies has been questioned and therefore these reports require confirmation (Phillips, Meredith, Spring & Chamberlin, 1982b). Control of ionic reabsorption from the locust rectum is better established. Using the voltage-clamped preparation developed by Williams, Phillips, Prince & Meredith (1978), Spring & Phillips (1980a,b,c; Phillips, Mordue, Meredith & Spring, 1980) demonstrated that a corpus cardiacum (CC) factor (chloride transport stimulating hormone, CTSH) acts via cAMP to stimulate active Cl- reabsorption across the rectal wall. The cellular mode of cAMP action has been extensively studied by Hanrahan & Phillips (1982, 1983a,b).
Goh & Phillips (1978) developed an everted rectal sac preparation which reabsorbs water and ions over a 5-6 h period in vitro at near constant rates which are close to those measured in vivo. They showed that water absorption in the absence of osmotic concentration differences and stimulants was coupled to active absorption of Cl-, and Na+ from the lumen. We therefore anticipated that CTSH, by increasing ion absorption, might enhance fluid transport; however, some preliminary results were inconclusive (Phillips et al. 1982b). However, Hanrahan (1982) in our laboratory subsequently observed that higher levels of cAMP (10 mm) are required to stimulate active Cl- reabsorption maximally in everted rectal sacs than in flat-sheet preparations (1 mm), presumably because of the small total amount of this cyclic nucleotide (e.g. 20 μA) usually placed inside these sacs. We were therefore encouraged to re-investigate the actions of cAMP and CC extracts containing CTSH on fluid absorption by everted rectal sacs, especially since we have recently elucidated the specific substrate requirements for rectal metabolism (Chamberlin & Phillips, 1982).
In this paper, we report evidence for an antidiuretic action of CTSH and cAMP. We also investigate the action of separated storage (NCC) and glandular (GCC) lobes of CC because Mordue (1970) has reported that an antidiuretic factor (ADH) is localized in the GCC, while a diuretic factor (DH) which inhibited fluid reabsorption is restricted to the NCC of Schistocerca and Locusta. The description of an antidiuretic role for CTSH, which is mostly (80 %) in the storage lobe, therefore appears to conflict with the results of Mordue (1970).
MATERIALS AND METHODS
The experimental animals were 30-day-old adult female Schistocerca gregaria, reared as previously described (Goh & Phillips, 1978) and starved overnight. Everted rectal sacs were prepared as described by Goh & Phillips (1978) except for the saline. Sacs were filled hourly with 10 (A of fresh saline and incubated in 25 ml of saline bubbled with 95% O2-5 % CO2 and maintained at a 25 °C. At hourly intervals for 4-5 h, weight gain and tissue volume change of rectal sacs were obtained by weighing recta before and after removing the fluid contained inside (i.e. on the haemocoel side; see Goh & Phillips, 1978). Correction for tissue volume change yields the true rate of transepithelial fluid movement. Student’s t-test was used to establish statistical difference between means. Entire corpora cardiaca (CC), or separated NCC and GCC of this gland, were removed from adult males to avoid cyclic changes associated with female reproduction. Each tissue was homogenized three times for 30 s (Dismembrator Artex, Sonic 300) in physiological saline and centrifuged for 30min at 24 000 (Sorval Superspeed R2CB Centrifuge). All these steps were carried out at 4 °C. Supernatants were stored frozen prior to assays, when they were tested in 10 /d of saline bathing the haemocoel side of rectal sacs. As a control tissue, 0-025 suboesophageal ganglion (SOG), which is several times the size of 0·5 CC, was treated in the same manner as the CC. Changes in osmolarity of the saline due to adding tissue homogenates were checked using an osmometer (Advanced Instrument Inc., Newton Highlands, Mass) and were found to be insignificant. Fresh CC or SOG extracts were added hourly to sacs after a 1-h equilibration period, unless otherwise stated.
The bathing saline (Phillips et al. 1982b) was based on ion, sugar and amino acid concentrations measured in locust haemolymph (Chamberlin, 1981; Hanrahad 1982). Osmotic concentration was adjusted to 427 mosmol I-1 with sucrose, which is not metabolized by locust rectum (Chamberlin, 1981): pH was 7-1. In some cases the osmotic concentration of this saline was raised to 827 mosmol I-1 with sucrose. A chloride-free saline was also prepared by replacing all chloride with nitrate. The stimulatory effect of cAMP was tested by including this agent in physiological saline on the haemocoel side.
Electropotential difference (PD) across the rectal wall was measured using two calomel electrodes in series with 3 M-KCl-agar bridges. The electrodes were connected to an high input impedance differential amplifier (1012Q, 4253 Teledyne Philbrick, Dedham, Mass.). The PD was measured at the end of each hourly incubation period prior to replacing the saline within the rectal sacs.
RESULTS
Stimulation of long-term fluid transport by CC extract and cAMP
The long-term absorption of fluid over the 2nd to 5th hour is shown in Fig. 1A. The rate of uptake (Jv) was 8·3 μl h-1 rectum-1 during the first hour (i.e. equilibration period, not shown) and dropped significantly (P< 0·025) to a steady-state level of 5 μl h-1 rectum-1 for control preparations containing 0·025 SOG. The volume of rectal tissue increased by 2-8 μl during the first hour, then decreased slightly (Fig. 1B). Control Jv values were not significantly different when saline lacking SOG was present inside the sacs for the whole 5-h period (data not shown). These results are close to those previously reported by Goh & Phillips (1978). Whole CC (0·5 glands) doubled long-term fluid absorption rate to 11 μlh-1 rectum-1 (Fig. 1A) compared with controls. Tissue volume changes were unaffected by CC extracts (Fig. 1B).
Initial measurements of fluid absorption at 30-min intervals indicated that CC extract exerted its maximum effect within the first 30min (data not shown). Moreover, replacing this saline with CC-free saline after 10 min did not diminish the subsequent stimulatory effect, suggesting that the active factor was already absorbed onto receptor sites by this time. Based on these results, hourly measurements of fluid absorption were judged appropriate to study the CC dose-response relationship (Fig. 2). There is a good linear relationship between the logarithm of the dosage added and the increase in fluid reabsorption over a range of 0·03 to 0·50 CC: higher CC dosages did not increase Jv further (data not shown but see Table 1). In contrast, the addition of 0-1 brain without pars intercerebralis (i.e. a considerably larger amount of nervous tissue than 0·5 CC) caused only a slight but insignificant increase in fluid absorption (Fig. 2).
Since cAMP is the apparent second messenger for CTSH (Spring & Phillips, 1980a) and mimics the action of CC extracts on KC1 absorption across shorted-circuited recta, we studied its effects on fluid absorption across everted sacs (Fig. 1). In comparison with controls, 10mm-cAMP caused an 80% increase in long-term fluid absorption which was significant at the end of the fourth hour. Jv increased linearly with the logarithm of cAMP concentration (Fig. 3), reaching a maximal value at 10 HIM-CAMP (i.e. in agreement with the results of Hanrahan, 1982, for stimulation Cl- transport across rectal sacs).
Measurements of PD across stimulated recta
All previous work with CTSH has been done on flat-sheet preparations of locust rectum. If CTSH, acting via cAMP, is the agent in CC extracts which stimulates fluid absorption across rectal sacs, we would expect a simultaneous two-to three-fold increase in transepithelial PD, because this hormone increases electrogenic Cl- transport and hence PD across flat-sheet preparations of recta bathed in low-K saline (Hanrahan, 1982). We measured both fluid absorption rate and PD on the same rectal sacs stimulated hourly with 0’5 CC for 4h. Recta absorbed 9·5 μl during the initial equilibration hour, after which the addition of CC supernatant caused a Jv of 11 μh-1 rectum-1 for the next 4h (i.e. twice control rates shown in Fig. 1). C extracts increased PD across these rectal sacs significantly (Fig. 4; P<0·01) from 1-5 mV (haemocoel side negative) to 35 mV, in quantitative agreement with previous results from flat-sheet preparations (Spring & Phillips, 1980a). In contrast, PD for unstimulated controls decreased slightly after the first h (Fig. 4). In summary, CC stimulation of Jv correlates with increases in PD due to CTSH stimulation of Cl- transport.
Cl− dependence of fluid reabsorption
If increases in Jv initiated by CC extract result from CTSH acting on rectal Cl- transport, stimulation of Jv should cease in Cl-free saline. Recta were bathed and fulled hourly with fresh Cl-free saline over a 4-h period. From the 3rd hour, half of These recta (the unstimulated group) were filled with Cl-free saline, while the remainder (stimulated group) received Cl-free saline containing 0·5 CC extract. The rates of fluid absorption are shown in Fig. 5A. The absence of Cl− in the bathing saline abolished the stimulatory action of CC extracts on fluid absorption, even at a high dosage of T2CC.
A complementary experiment is shown in Fig. 5B. After a 2-h period during which recta were bathed and filled with Cl-free saline, they were re-exposed to normal saline (i.e. with Cl-) with or without 0-5 CC. After a decrease in absorption rate due to the lack of Cl- during the first 2h, the absorption rate increased significantly when Cl− was re-added. However this increase was much greater (two-fold) in recta exposed to CC extract (P < 0 ·001 ). Clearly, stimulation of fluid transport by CC extract specifically requires external Cl-, as expected if CTSH is the stimulatory agent. Unstimulated recta are still able to transport fluid at low rates without exogenous Cl-, because under these conditions cation transport is known to sustain some fluid uptake (Phillips et al. 1982b).
Is the factor stimulating fluid absorption located in both NCC and GCC, as is CTSH (Phillips et al. 1980), or only in the GCC with an inhibitory factor (DH) in the NCC, as reported by Mordue (1970)? Supernatants of NCC and GCC removed from the same animals were added hourly to everted sacs for 4 h at a dose of either 0·125 or 1·00 gland-equivalents in 10 μl. The averaged hourly fluid absorption rates are shown in Table 1. Both NCC and GCC caused a significant increase in fluid absorption at both dosages (P< 0·025 and P< 0·001 respectively). These results are consistent with stimulation of fluid absorption by CTSH, but not with an inhibitory DH located in the NCC and an ADH only in the GCC (Mordue, 1970).
Fluid absorption against osmotic gradients
We have so far only considered Jv under isosmotic conditions. Phillips (1964) showed in vivo that Jv was linearly dependent on the osmotic concentration difference (AOsm) across the rectal wall. Goh & Phillips (1978) confirmed this in vitro using unstimulated everted sacs. Because Jv/AOsm indicates the osmotic permeability Posm) of the rectal wall, the effect of CC extracts on this relationship should indicate whether active absorption of fluid (driven by Cl- transport) or passive osmotic permeability of the rectal wall are changed. We measured Jv hourly for recta bathed externally in a hyperosmotic saline (827 mosmolT1) and filled hourly with 10/d of isosmotic saline containing 0·025 SOG (control), 1NCC, or 1GCC. This 400 mosmol I-1 difference provoked a dramatic decrease in Jv in all three experimental groups, but especially in the control group where the direction of fluid movement actually reversed (Fig. 6). During the whole 4-h experiment, Jv of the control group remained significantly lower than that of the stimulated groups (P< 0·001 or P< 0·005). Clearly, NCC and GCC containing CTSH cause recta to absorb against much larger AOsm than do unstimulated controls. The increases in Jv caused by CC extracts were the same (4·5 μlh-1 rectum-1) when AOsm across the rectal wall was 0 (Fig. 1) or 400 (Fig. 6), indicating no change in P0Sm. This suggests that the stimulant in CC acts on ion-driven active fluid absorption (as expected for CTSH), which should increase the AOsm against which fluid absorption can occur: increased Posm would have had the opposite effect.
DISCUSSION
It was previously not possible to conclude whether CTSH acts as an ADH to stimulate fluid absorption, or strictly as an ‘aldosterone-like’ hormone which only increases KC1 transport (Phillips et al. 1982Ô). The new supportive evidence that CTSH does act as an antidiuretic factor on locust rectum is as follows: (1) the neuroendocrine tissue (CC) containing CTSH doubles the rate of fluid transport whereas larger amounts of other nervous tissue (e.g. SOG) have no effect. (2) The demonstration of approximately equal amounts of antidiuretic activity in both storage and glandular lobes of CC (Table 1) is qualitatively consistent with the location of CTSH activity determined by increases in Cl-dependent short-circuit current (Isc; Phillips et al. 1980), but different from the distribution of other CC factors reported to date. (3) The dose-response curve for CC stimulation of fluid absorption (Fig. 2; maximum rate with 0-5 CC) is in reasonable agreement with the relationship for CC stimulation of Isc, as recently measured in our laboratory (i.e. maximum Isc obtained with 0-3 CC; Hanrahan, 1982; J. Meredith & J. Proux, unpublished observations). Spring & Phillips (1980a) reported a somewhat more sensitive response of rectal Isc to CC (i.e. 0·1 gland giving maximum Isc). (4) Cyclic AMP, which mimics CTSH stimulation of KC1 absorption, also stimulates fluid transport by rectal sacs (Fig. 1 A). (5) CC extract causes an increase in transepithelial PD concurrent with the increase in fluid transport across rectal sacs, as predicted if increased fluid movement resulted from stimulation of electrogenic Cl- transport by CTSH. (6) In support of this interpretation, the antidiuretic action of CC extracts on Jv is abolished when recta are bathed in Cl-free saline (Fig. 5), even though we have shown that NaNCh and KNO3 (i.e. Cl-free) solutions can sustain some fluid transport by unstimulated recta (Phillips et al. Clearly the increase in rectal fluid transport caused by CC extract is specifically associated with stimulation of Cl- transport, which is also the known action of CTSH. (7) Finally, CC extracts stimulate the active ion-dependen component of fluid absorption across locust rectal sacs against larger osmotic gradients, rather than the passive component (i.e. osmotic permeability or back diffusion, Fig. 6). This is again consistent with CTSH being the active agent. In summary, there is considerable evidence that CTSH is probably the effective stimulant responsible for the antidiuretic actions of CC extracts reported in this paper. We have run a preliminary test of the antidiuretic activity of purified CTSH by HPLC: in spite of rapid loss in biological activity, this preliminary test was positive.
The results in this paper are for long-term transfer of fluid across well-characterized rectal preparations, excluding data for the first hour when rapid decreases in fluid and ion absorption rates and large tissue volume changes are known to occur (Goh & Phillips, 1978; Phillips et al. 1982b; Williams et al. 1978; Fig. 1 of this study). Most earlier studies on the control of fluid absorption across insect recta were carried out on uncharacterized rectal preparations, often during the initial transient phase and without knowledge of specific incubation conditions required to sustain prolonged fluid absorption (see Phillips et al. 1982b). For example, Mordue (1970) measured fluid absorption over a 1-h period using everted rectal sacs of locusts bathed in a simple saline without oxygenation or the required metabolic substrate, proline. He reported that the NCC inhibited rectal fluid absorption (diuretic effect), whereas we consistently observed an antidiuretic effect of this tissue on long-term fluid transport. We are in agreement, however, that the GCC has an antidiuretic action on everted rectal sacs of locusts, although properties of Mordue’s ADH factor and CTSH appear to be different (see Phillips et al. 1980). Since the DH reported in CC by Mordue is unstable (see Morgan & Mordue, 1983), this factor may have been inactive in our extracts. The VP-like DH in locust SOG (Proux, Rougon & Cupo, 1982) apparently does not influence rectal Jv, although we have still to test this in the presence of CTSH.
There is some evidence that CTSH, like DH, is released after feeding (Phillips et al. 1982a). As a working hypothesis, we therefore suggest that the combined action of these two hormones might be to increase recycling of KCl-rich fluid through the excretory system. This would serve to clear the body of waste substances ingested with or produced by metabolism of the meal. At the same time, changes in relative levels of DH and CTSH with time would ensure that excess fluid is eliminated quickly.
ACKNOWLEDGEMENTS
This research was supported by operating grants from NSERC, Canada.