Mechanisms responsible for maintaining internal stability within organisms are referred to as homeostatic mechanisms. Homeostasis is the maintenance of static conditions in the internal environment. Homeostatic mechanisms are active processes because conditions are nearly always subject for forces that will bring about changes. Like virtually all other organisms, terrestrial and aquatic insects confront the problem of maintaining an optimal balance of water and of salts between the intracellular and extracellular compartments. With respect to insects, the most general physiological expression is that ions and water are secreted by Malpighian tubules and they are selectively resorbed in hindguts. Osmoregulation is a complex phenomenon involving mechanisms to reduce and offset water losses, and mechanisms to recover optimal osmotic balances when they are insulted.

Removing excess water from the extracellular compartment is known as diuresis. Diuresis is regulated at the organismal level by the actions of diuretic hormone (DH) and antidiuretic hormone (ADH). (Take a look at this process) Most of our information on the regulation of diuresis by DH and ADH comes from research on adult mosquitoes, on the blood-sucking bug Rhodnius and, of course, on the locust. Hematophagous insects experience a challenging insult to the homeostasis of water and salt balance each time they take a blood meal. Females of the yellow fever mosquito, for example, often consume twice their body weight in a single blood meal. The vast majority of the weight is water. Imagine, for a moment, what it might feel like to eat about 50 kg of watermelon in less than 5 minutes. Female mosquitoes often begin diuresis within about 2 minutes of starting a blood meal, even before they complete the meal. The rapid diuretic response occurs in response to DH. DH is released from the brain, by way of median neurosecretory cells. The hormone stimulates increased fluid secretion rates in the Malpighian tubules. The diuretic response results in a return to an appropriate balance of salts and water between the intracellular and extracellular compartments.

It might be thought that DHs occur in those insects that are subject to extreme challenges to osmotic homeostasis, but not in other species. This is not the case. Endocrine regulation of Malpighian tubule function was first described in locusts over 20 years ago. The DH activity was found in extracts of the corpora cardiaca. When the extracts were separated by high performance liquid chromatography, two separate peaks were detected. The peptides associated with both peaks were concentrated, and tested for diuretic activity by bioassay. Both peptides stimulated fluid secretion in Malpighian tubules. Similar extracts of other tissues, including brain, sub-esophageal ganglion, and thoracic ganglion all stimulated increased fluid secretion. Extracts from the brain had two active peaks, but all the other ganglia had a single peak of activity, then known as DIURETIC PEPTIDE I.

Mammals also produce DHs, one of which is known as arginine vasopressin, or AVP. A group in Bordeaux, headed by Jacque Proux, used antibody antibodies from a commercial radioimmunoassay kit for AVP to detect a factor in extracts of the sub-esophageal ganglion. The factor was called AVP-like DH due to its antigenic similarities to mammalian AVP. This group recently isolated the AVP-like DH from thoracic and sub-esophageal ganglia. Their work makes an interesting story. The group began by removing the glands from insects, then storing them in acetone. Over a period of some years, they collected a total of 51,000 glands.

The glands were homogenized, and the DH was prepurified by centrifuging the homogenate at 100,000 X g for 1 hour, then filtering the supernatant on silica gel cartridges. The next step was semi-purification by HPLC on a Vydac column. As it turned out, most of the immunoreactivity was associated with the first of the two peaks, but all of the biological activity on actual Malpighian tubules was in the second fraction.

Amino acid studies showed that both factors had the same amino acid composition. The factors were then separated on size exclusion chromatography. This indicated that the first factor had a molecular weight of about 700 daltons and the second factor was about 1400 daltons. This finding suggested that the biologically active factor was a dimer of the first factor. It is not at all unusual to have two peptide chains hooked up to form a single functional entity. The mammalian hormone, insulin, is a classic example. Very often the chains are connected at cysteine residues by disulfide bridges. The next step was to obtain the amino acid sequence of the first factor. The issue then became a matter of learning how the peptides are connected. They could be connected in a parallel or anti-parallel form. An extremely talented peptide chemist named David Schooley synthesized both forms. Only the anti-parallel dimer had biological activity on Malpighian tubules. The AVP-like DH of Locusta is an anti-parallel dimer. This is the first insect DH to be isolated, sequenced and synthesized.

DHs have been detected in aquatic insects. A factor extracted from heads of the water boatman Cenocorixa blaisdelli caused an 8-fold increase in fluid secretion rates. Similarly, extracts of thoracic ganglion caused an increase in fluid secretion rates in larvae of the dragonfly Libellula quadrimaculata.

Several terrestrial insects, including the house cricket Acheta domesticus also produce a DH.

It is generally thought that DHs regulate activity in Malpighian tubules and in hindguts. We can model the hormone actions in a figure.

The figure indicates that factors from the storage lobes of corpora cardiaca cause increased fluid secretion rates in Malpighian tubules and decreased water resorption in hindguts. These actions are consistent with eliminating water from the extracellular compartment at the organismal level. Eliminating water would be a diuretic action.

For many years the received wisdom was ADHs appear to act on hindgut, but not on Malpighian tubules. However, more recently we have learned that ADH factors also act on the Malpighian tubules. ADH appears to increase water resorption rates in isolated recta, and decrease water secretion in Malpighian tubules. Actions at both tissues constitute ADH action.

It is believed that biological epithelia can not directly pump water from one side of the membrane to another. The general expression is that ions can be selectively moved, then water follows a local gradient. If this is so, then how can water be selective moved out of the rectum, leaving salts behind? We consider a model. The model is a schematic of the principal cells of rectal pads in cockroaches and locusts. These cells are characterized by scalariform complexes and intercellular spaces, which are quite rigid. Active transport processes are thought to create very high osmotic concentrations within the rigid scalariform complexes. Water in the lumen of the rectum would then move into these hyperosmotic spaces. These spaces are rigid, so the osmotic movement increases hydrostatic pressure. The increased hydrostatic pressure forces water to move through the spaces, and finally into the hemocoel. The final openings to the hemocoel are thought to be valve-like to prevent flow in the reverse direction. According to this model, water is moved from one side of a membrane to the other. Here, we emphasize the water movement is powered by the usual physiological actions: ions are pumped, and water follows a local osmotic gradient. The key point is the rigid structures within the cells create a directional water movement.

The model has been supported by electron-probe X-ray micro-analysis. This kind of analysis is performed on a scanning electron microscope. A beam of electrons is focussed on a section of the cell. Ions within the cell release X-rays which are characteristic of the particular ion. The characteristic X-rays are detected by photomultiplier tubes.

The movement of water is regulated by moving ions within principal cells of the recta. The ADH of insects may be identical to a hormone known as chloride transport simulating hormone (CTSH). This is also a factor that has been isolated from corpora cardiaca of locusts. CTSH has been shown to cause electrogenic chloride movement within recta.

Physiological models are useful tools. Nonetheless, we want to underscore the point that these are models. Models are continuously changed to accommodate new information. Let us revisit our general model of DH and ADH actions. As mentioned just above, another anti-diuretic factor has been isolated from the corpora cardiaca, in this case during work on the house cricket. This factor acts on Malpighian tubules and causes a reduction in fluid secretion. Hence, in the final analysis, we might come to the view that DHs and ADHs act on Malpighian tubules and recta in coordinated ways.

As we've just seen in insects, mammalian kidney function is regulated by major hormones. However, other factors also exert regulatory actions. One is a class of molecules known as eicosanoids. These molecules are synthesized from certain polyunsaturated fatty acids. David Petzel and I developed the hypothesis that eicosanoids might also be important elements in regulation of Malpighian tubule physiology. We tested this idea in direct studies of mosquito Malpighian tubule function. We treated Malpighian tubules with eicosanoid biosynthesis inhibitors, then observing the effects of the inhibition on fluid secretion rates in adult females of the mosquito Aedes aegypti. We found that increasing doses of indomethacin (a prostaglandin biosynthesis inhibitor) resulted in decreases in fluid secretion rates. Our current idea is that the prostaglandins modulate the effects of major hormones at the tissue level. It follows that there are at least two tiers, or levels, of regulation of fluid secretion. Future studies will probably make the picture even more complicated.

Malpighian tubule and hindgut functions involve moving ions from place to place. We use the expression epithelial transport to describe the movement of ions from hemolymph into Malpighian tubule lumen or from rectal lumen into hemolymph. Epithelial transport entails moving ions across one cell membrane, through the cell, and finally across another cell membrane. Epithelial transport occurs through a number of processes. One process is movement through ion channels. An ion channel might look like a couple of large proteins assembled such that they form a tube running through a biomembrane. We also can imagine a group of proteins forming a tube or channel allowing passive movement of ions according to the forces of electrochemical gradients. The channels are of different sizes, and it is the size differences that determine movement of specific ions.

Ions can be moved across epithelia against electrochemical gradients by active processes. Primary active transport processes directly the potential energy in ATP to move ions. Examples of active ion pumps include the sodium-potassium ATPase, and the sodium-potassium-2chloride co-transporters. The exact shape and operation of these pumps are unknown, but it is thought that the transporter proteins undergo a change in conformation with the expenditure of energy, and that the altered conformations physically move the ions from one side of a membrane to another.

People have tried to model the sodium-potassium ATPase actions. In the first step, sodium binds to the protein. The protein is then phosphorylated by hydrolysis of ATP. Sodium binding and phosphorylation, both on the cytoplasmic side of the protein, cause a conformational change resulting in transporting the sodium to the other side of the membrane, where it can be released into the medium. After releasing the sodium, the protein is available to bind potassium. Potassium binding leads to dephosphorylation of the ATPase, which returns the protein to its dephosphorylated conformation. The return transports the potassium ion across the membrane. The potassium ion is released from the ATPase, and the transport cycle can begin again. This model suggests that one sodium and one potassium ion are transported in each phosphorylation cycle. However, the biological sodium-potassium ATPase has three sodium binding sites and two potassium binding sites.

Let's look at another biological significance of secretion in insect systems. Increased Malpighian tubule and rectal fluid secretion rates is thought to serve a function that goes beyond homeostasis of salt and water balance in insects. It has been proposed that DH and ADH could work in concert in increase fluid cycling with the body. The increased fluid cycling is thought to facilitate filtration of metabolic wastes and xenobiotic chemicals from the hemolymph.