Lk

Figure 4.6 Ion transport pathways in Malpighian tubules of (a) Drosophila and (b) Aedes. Note: Diagrams illustrate the different ways in which kinins are thought to stimulate the movement of Cl~ ions. In Drosophila, leucokinin (LK) acts via Ca2 + to modulate Cl~ channels in the stellate cells (hatched). In Aedes, LK enhances Cl~ transport through the paracellular pathway.

Source: Reprinted from Journal of Insect Physiology, 46, O'Donnell and Spring, 107-117, © 2000, with permission from Elsevier.

cation pump' is thus equivalent to the parallel operation of proton pump and antiporters. At the basolateral membrane, routes for K+ and Na+ entry include channels, Na+: K+: 2Cl~ or K+: Cl_ cotransporters, and the Na +/K+-ATPase, their relative importance differing according to insect species (Leyssens et al. 1994; Linton and O'Donnell

1999). Electrical coupling between the two membranes serves to balance basolateral entry of cations and their apical secretion (Beyenbach et al.

2000). The apical H+ -ATPase generates a favourable lumen-positive transepithelial potential for counter-ion, predominantly CP, transport. Water movement across the tubule is considered to be osmotically coupled to ion transport and ultrastructural observations suggest that it is transcellular (O'Donnell 1997).

Anion transport in Malpighian tubules is less well understood than that of cations, and there is controversy concerning the pathway of passive Cl_ transport, which is considered to be transcel-lular in Drosophila (O'Donnell et al. 1996, 1998) but paracellular in Aedes (Pannabecker et al. 1993). Tubules of both species contain principal cells interspersed with much smaller stellate cells, and in Drosophila peptides of the kinin family are thought to open Cl~ channels in stellate cells via an increase in intracellular Ca2 + (O'Donnell et al. 1998). Even though CP channels have recently been identified in stellate cells of Aedes tubules (O'Connor and Beyenbach 2001), fast fluid secretion may still involve a paracellular shunt pathway for CP, with stimulation by kinins changing the tubule from a tight to a leaky epithelium

(Beyenbach 1995). In contrast, tubules of the tsetse fly Glossina morsitans (Diptera, Glossinidae) are permeable to Cl~ ions even before stimulation (Isaacson and Nicolson 1994).

Malpighian tubules of Drosophila melanogaster have become a valuable model in molecular physiology (Dow et al. 1998; Dow and Davies 2001). Only 2 mm long, with 145 principal cells, these tiny tubules can be studied by standard techniques for measuring fluid secretion rates and membrane potentials, but possess unique advantages as an experimental model because of the genetic tools available for this organism. For example, complex heterogeneity of Malpighian tubules (previously underestimated in this epithelium and others) has been demonstrated by the mapping of physiological domains to the level of single cells (Sozen et al. 1997). Cloning and characterization of several V-ATPase subunits have provided the first gene knockouts of V-ATPases in an animal (Dow et al. 1997).

Malpighian tubules: hormonal control Fluid secretion by Malpighian tubules is stimulated, often dramatically, by diuretic hormones. These were initially discovered in blood-sucking insects which experience severe osmotic challenges which are corrected with a fast post-prandial diuresis (Maddrell 1991). Diuretic hormones are apparently present in all insects, including those with far slower rates of water turnover such as desert beetles (Nicolson and Hanrahan 1986): in the latter the function of such hormones may be clearance of the haemolymph with recycling of water (Nicolson 1991). The first corticotropin releasing factor (CRF)-related diuretic peptide was characterized from adult heads of the tobacco hormworm Manduca sexta by Kataoka et al. (1989). The majority of diuretic hormones characterized to date belong to two neuropeptide families: the CRF-related peptides and the smaller kinins (for review see Coast 1996; Coast et al. 2002). The CRF-related peptides share various degrees of homology with vertebrate CRF and act through the second messenger cyclic AMP to increase the rate of cation transport (Beyenbach 1995; O'Donnell et al. 1996). The kinins, initially isolated on the basis of myotropic activity on hindgut preparations, act independently through an increase in intracellular Ca2 + concentration to increase anion permeability of Malpighian tubules (O'Donnell et al. 1998; Yu and Beyenbach 2001). This separate control of cation and anion transport suggests the possibility of synergistic control of secretion by CRF-related peptides and kinins, and this has been demonstrated in Malpighian tubules of the locust Locusta migratoria (Fig. 4.7) and the house fly Musca domestica (Coast 1995; Iaboni et al. 1998). Co-localization of kinins and CRF-related peptides in neurosecretory cells would ensure their coordinated release to regulate fluid secretion (Chen et al. 1994).

Tubules of Rhodnius prolixus (Hemiptera, Reduviidae) show synergism between serotonin, which acts as a diuretic hormone after a blood meal in this species, and forskolin, which stimulates adenylyl cyclase and mimics the effect of the CRF-like diuretic peptide (Maddrell et al. 1993). Such synergism achieves a more rapid response using smaller quantities of peptides. Other insect diuretic hormones include calcitonin-like peptides (Furuya et al. 2000), and cardioactive peptide 2b (Davies et al. 1995). Recently, an antidiuretic peptide which uses cyclic GMP as second messenger and inhibits tubule secretion has been isolated from Tenebrio

-2-10 1 2 Log concentration (nM)

Figure 4.7 Synergistic stimulation of Malpighian tubule secretion by CRF-related peptides and kinins. Locust diuretic peptide (squares) and locustakinin (circles) were assayed separately and together (triangles) on Malpighian tubules of Locusta migratoria. Means ± SE (n = 5 to 8).

-2-10 1 2 Log concentration (nM)

Figure 4.7 Synergistic stimulation of Malpighian tubule secretion by CRF-related peptides and kinins. Locust diuretic peptide (squares) and locustakinin (circles) were assayed separately and together (triangles) on Malpighian tubules of Locusta migratoria. Means ± SE (n = 5 to 8).

Source: Reprinted from Regulatory Peptides, 57, Coast, 283-296, © 1995, with permission from Elsevier.

molitor (Eigenheer et al. 2002). The complexity of control of insect Malpighian tubules was discussed by O'Donnell and Spring (2000), and their ideas are supported by studies of the antagonistic action of synthetic endogenous diuretic and antidiuretic peptides in Tenebrio tubules (Wiehart et al. 2002), and by the multiple diuretic factors which seem to be present in many insects (for a definitive review see Coast et al. 2002). The latter authors stress that physiological relevance in vivo is much more difficult to demonstrate than the cellular actions of identified diuretic or antidiuretic factors. As yet, few studies meet the stringent criteria (e.g. haemolymph titres of appropriate timing and concentration) for these factors to be assigned functional roles as neurohormones.

Hindgut

The reabsorptive regions of the insect excretory system consist of an anterior ileum and posterior rectum (structurally more complex), and the transport mechanisms involved and their control have been reviewed by Phillips and colleagues (Phillips et al. 1986, 1998), mostly based on the best studied insect hindgut, that of the desert locust S. gregaria. For other insects, the mechanisms and the control are less well known.

Malpighian tubules enter the gut at the midgut-hindgut junction, and the hindgut is lined with cuticle which protects the tissue and acts as a molecular sieve (Phillips et al. 1986). Primary urine first passes through the ileum, which in locusts appears to be functionally analogous to the mammalian proximal tubule, because it is responsible for bulk isosmotic reabsorption of primary urine. Studies of the ileum, using well characterized in vitro preparations initially developed for the rectum (Hanrahan et al. 1984), have shown that the driving force for fluid reabsorption is electrogenic CP transport across the apical membrane. The counter-ion is K + (generally present at high concentration in the primary urine) and the ileum is also the site of reabsorption of Na + (Irvine et al. 1988).

The insect rectum is analogous to the more distal segments of the mammalian nephron (Phillips et al. 1996a). Structural modifications for reabsorption are seen in rectal pads or papillae, derived from a single layer of columnar cells. Current ideas concerning the mechanism of fluid reabsorption are based on ultrastructural studies and micropuncture sampling from various compartments, supported by microprobe analysis of ion distribution (Gupta et al. 1980). These techniques show that, as in other epithelia, ion pumping creates osmotic gradients and ultrastructural details lead to net water movement (reviewed by Phillips et al. 1986). Ion transport into narrow intercellular spaces (Fig. 4.8) creates locally high osmolalities, so that water

Osmotically driven

H2O H2O

Cuticle

Apical microvilli^

Mitochondria Septate junction

Septate junction Tight junction

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