Introductory Discussion on Water, pH, Buffers, and General Features of Receptors, Channels, and Pumps

Diabetes Insipidus

Failure of the kidneys to reabsorb water and concentrate the urine results in greatly diluted urine and substantial loss of water from the body. There are two causes for this condition, undersecretion of the posterior pituitary hormone, arginine vasopressin ( AVP or ADH, a nti d iuretic h ormone), or failure of the distal tubule of the kidney to respond to AVP. Failure of the kidney to respond to AVP can be a function of the loss of function of the AVP receptors (AVPRs) or the loss of function of the water channels [aquaporins (AQPs)] required for the reabsorption of water from the urine. Undersecretion of AVP from the posterior pituitary is called central diabetes insipidus , and failure of the kidney to respond to AVP is called nephrogenic diabetes insipidus . Central diabetes insipidus is rare accounting for 1 person in 25,000. Nephrogenic diabetes insipidus can occur with the abuse of alcohol or some types of drugs and with kidney disease, sometimes associated with the aging process. Substantially, smaller amounts of cellular proteins are synthesized in cells during the aging process and, consequently, there may be some loss of the receptor for ADH in the kidney. Moreover, there may be loss of function mutations of the genes for the AVP precursor ( preprovasopressin ), the genes for the AVPR, or the genes for AQPs. Excessive urination and loss of water also occur in untreated diabetes mellitus (due to the loss of insulin by destruction or underactivity of the beta cells of the pancreas or due to insulin resistance). In this case the loss of water is related to high levels of glucose in the blood and urine. Glucose carries bound water with it ( Fig. 3.1 ) in the process of excretion, a completely different causation than the factors in diabetes insipidus .

Water is a dipolar molecule, wherein the oxygen has a large nucleus that attracts electrons causing the oxygen atom to be negatively charged, whereas the hydrogens are positively charged. This dipolar character allows water molecules to interact with each other through hydrogen bonding ( Fig. 3.2 ).

Sugar hydroxyl groups are attracted to the oxygens of water to form hydrogen bonds and maintain the sugar in solution. Anions (negatively charged atoms or molecules that would be attracted to the positively charged anode during electrophoresis) will have strong interactions with water. The same is true for cations (atoms or molecules with a positive charge that would be attracted to the negatively charged cathode during electrophoresis). For example, a sodium ion (cation) will attract six molecules of water as shown in Fig. 3.3 .

The determination of the number of water molecules interacting with a cation (Na ⁺ ) is complex and depends on a number of variants solved by the formula, [M(H ₂ O) n ] z +, where M is the metal ion, z ⁺ is the electrical charge, and n is the number of interacting water molecules. An important factor in the determination of the number of interacting water molecules is the radius of the ion where the interaction is greater with a smaller radius (Na ⁺ has a small radius).

Thirst and Arginine Vasopressin

Diabetes insipidus is also characterized by intense thirst . Both thirst and the mechanism to release AVP are interrelated through neuronal connections in the hypothalamus . AVP is cut out of its precursor, preprovasopressin ( Fig. 3.4 ).

AVP is synthesized in vasopressinergic (releasing AVP) neurons located in the supraoptic and paraventricular nuclei of the anterior hypothalamus. AVP is transported in combination with a neurophysin ( NPII ) protein down the long axon to the neuronal terminal located in the posterior pituitary. It is secreted in response to increased osmolality (an increase in the solute particles in plasma but usually an increase in sodium-ion concentration) from the neuronal terminal by a process of exocytosis activated by increased calcium ion concentration. AVP is secreted when the osmolality of the plasma is increased by only 1%–2%. It appears that the TRPV channel (transient receptor potential channel) family of cation channels may be involved as osmomechanical receptors . Possibly, they could mediate neuronal responses to changes in tonicity involving the changes primarily in Na ⁺ concentrations in plasma. For example, the TRPV4 channel is a Ca ²⁺ -permeable cation channel that is involved in the regulation of systemic osmotic pressure. AVP has a relatively short half-life of about 15 minutes as it is converted in the liver and kidney to inactive products.

AVP has the following amino acid sequence:

with a disulfide bridge connecting the two cysteine (C) residues. This hormone is similar to oxytocin, another posterior pituitary hormone. Oxytocin is also a nine–amino acid peptide having the same sequence except that amino acid 3 (phenylalanine, F) is replaced by isoleucine (I), and amino acid 8 (arginine, R) is replaced by leucine. Both hormones have identical disulfide bridges connecting to the cysteines.

There are four major stimuli leading to thirst . As already described, hypertonicity , particularly an increase in the Na ⁺ concentration in plasma, operates through an osmoreceptor in the hypothalamus. Decreased volume of water, hypovolemia , is sensed by low-pressure baroreceptors located in the large veins and in the right atrium of the heart. Lowered blood pressure, hypotension , is sensed through high-pressure baroreceptors located in the carotid sinus and aorta. In decreased water volume the consequently increased osmolality in the extracellular fluid will lead to the decreased secretion of saliva that, in turn, leads to dry mouth , creating the sensation of thirst. Some cases of migraine headache may be the result of inadequate hydration. Finally, the hormone, angiotensin II (ANGII) , is produced through the action of the enzyme renin secreted from the kidney in response to renal hypotension . ANGII binds and activates specific osmoreceptors located in the subfornical organ (SFO; in the lower fornix) and in the organum vasculosum of the lamina terminalis . These are connected to the median preoptic nucleus of the hypothalamus ( Fig. 3.5 ). The action of AII also leads to thirst.

ANGII acts as a neurotransmitter in the neuronal path from SFO to the hypothalamus. In the same area of the hypothalamus, there are ascending pathways from low- and high-pressure baroreceptors that signal thirst development. It is believed that the osmoreceptor stimulating release of AVP and the receptors stimulating thirst are located in the same region of the hypothalamus . The overall picture for the development of thirst and the roles of angiotensin II are summarized in Fig. 3.6 .

Action of Arginine Vasopressin on the Distal Kidney Tubule

The molecular actions of AVP in the kidney are discussed in the chapter on polypeptide hormones. Here, only a brief description will be given. After the AVP-neurophysin II, complex is released from the posterior pituitary into the general circulation, it reaches the vicinity of AVPR. AVPR is located in the membranes of cells of the distal convoluted tubule and the collecting ducts of the kidney. In the vicinity of the AVPR, neurophysin II is dissociated from the complex so that free AVP can bind and activate its receptor. AVPR is a G protein–coupled receptor and, through its action, cyclic adenosine monophosphate (cAMP) is formed from adenosine triphosphate (ATP). cAMP activates protein kinase A (PKA) that phosphorylates microtubular subunits . The phosphorylated microtubular subunits form AQP channels that insert into the apical cell membrane. These channels allow the entry of water so that the water in vesicles can be transported across the cell to the basolateral membrane where the water is released into the extracellular space and finally into the bloodstream to increase its volume.

Water and Biological Roles

Water is a key substance for life. While the human body, depending on its condition and many other factors, can live for weeks, even months without food, one will usually die if deprived of water for 3 days. The human body is composed of about 72%–78% water. Of this, about 70% is intracellular and 30% is extracellular. The water molecule is unique; it has polarity with oxygen bearing a negative charge at one end and two hydrogens bearing positive charges at the other end ( Fig. 3.7 ).

Water is the major solvent in the body for all kinds of nutrients, salts, intermediate compounds, and others. It is critical for the activity of proteins ( Fig. 3.8 ).

The Photocatalytic Dissociation of the Water Molecule Into Its Gaseous Atoms

The reaction: H ₂ O=H ₂ +1/2O ₂ Δ G ⁰ =237.13 kJ/mol (where G is the Gibbs free energy; J=Joule) is one that is carried out in green plant photosynthesis . Chlorophyll , a structure similar to heme in mammalian blood ( Fig. 3.9 ), in green plants, captures photons from sunlight; chlorophyll becomes “activated” allowing one of its electrons to be transferred to another molecule. Chloroplasts (subcellular particles containing chlorophyll in which photosynthesis takes place) are able to convert water into oxygen (and hydrogen gas) and carbon dioxide into carbohydrates needed for plant growth ( Fig. 3.10 , ). The overall equation for light-dependent photosynthesis is 2H ₂ O+2NADP ⁺ +3ADP+3P _i =O ₂ +2NADPH [ electron transport chain+Calvin cycle+CO ₂ = 3C sugars ]+3ATP; [chlorophyll+photon=chlorophyll* ( excited state )−photosystem II-electron transport chain+ H ⁺ ]. When chloroplasts are isolated from plant cells, they are still capable of dissociating water into oxygen and hydrogen but are unable to convert carbon dioxide into carbohydrates (biochemical reactions in carbohydrate synthesis are conducted on a different membrane). Modern chemical research endeavors to simulate the energy-requiring dissociation of water into its component gases to produce a readily transportable fuel as H ₂ and increase the level of oxygen in the atmosphere without any detrimental side effects. A recent review summarizes the ongoing research on this problem ( ). Although the chemical system is demanding (endogenous water must be removed from the system; the development of suitable metal photocatalysts is demanding, etc.), progress has been made so that a further increase in the efficiency of the process and its increasing economy will make it applicable to large scale through which portable fuel (as H ₂ ) can be produced with accompanying enrichment of the atmosphere in terms of the addition of oxygen. Fig. 3.11 shows the two versions of the process ( ). In Fig. 3.11A a single artificial photocatalyst can capture visible light and convert water into oxygen and 2H ⁺ into H ₂ . In Fig. 3.11B the same process is accomplished except that two artificial photocatalysts are involved, one for the conversion of water into O ₂ and the second for the conversion of 2H ⁺ into H ₂ (there have been a number of artificial photocatalysts developed, one of which is MgTa ₂ O _{6− x} N _y /TaON (Ta is tantalum), where the photocatalyst is loaded with Pt by impregnation and subsequent H ₂ reduction method). In view of the limitless supply of water on earth, especially if inexpensive desalinization can be accomplished, this development could solve humanity’s limiting availability of sources of energy.

Water Channels: Aquaporins

Water is not able to cross the nonpolar cell membrane because water is insoluble in lipids, whereas lipid molecules often can dissolve in the lipophilic cell membrane and freely diffuse into the cell. The special mechanism by which water enters a cell is a water channel , called an AQP , discovered in the 1980s. AQPs occur in most cell types, including the red blood cell. Because of the nature of the water reabsorption process in the kidney, it has been revealed that there are at least four different AQPs. There are two apical AQPs (AQP1 and AQP2) for water reabsorption; the second one operating in response to ADH (or AVP). The other two AQPs (AQP3 and AQP4) in the kidney operate basolaterally for water reabsorption. In the human body, at least 13 different AQP protein variants have been identified starting with AQP0 through AQP12 . AQP1 from the red blood cell is the best studied ( Table 3.1 ). AQP4 is highly expressed at the blood–brain barrier in the end feet where it regulates water homeostasis and the formation of cerebrospinal fluid. When water accumulates in the brain ( edema ), it appears that AQP9 is involved. Experimental observations suggest that AQP4 may play a role in fluid elimination rather than edema formation after brain injury.

A simplified drawing of an AQP water channel is shown in Fig. 3.12 . It has been predicted that the AQP1 channel could facilitate the transport of water at a theoretical rate of about 3 billion water molecules per second ( http://www.bio.miami.edu/~cmallery/150/memb/water.channels.htm ). One group of AQPs is impermeable to charged molecules or protons but able to admit some other small molecules, such as glycerol ( aquaglyceroporins ).

Fig. 3.12 shows that the water molecules on the apical side of the cell enter the channel with the oxygen atom facing downward and the two hydrogen atoms facing upward. As water proceeds downward, single file, midstream through the channel, the water molecule inverts so that the same has the two hydrogen atoms facing downward and the oxygen atom facing upward. This controlled inversion is mediated by the side chains of amino acids of the channel as the water molecule proceeds through it. Asparagines , hydrogen bonding to the oxygen of water, apparently make this inversion possible (some publications question this exact version of the flow of water molecules and also question the exclusion of proton flow through the AQP channel). AQPs can conduct water in both directions, in and out of the cell. The inward action takes place at the apical membrane and the outward action at the basolateral membrane (facing the extracellular space and ultimately the bloodstream). When water leaves a cell through an AQP channel, it leaves with the oxygen atom facing upward. In a kidney cell, for example, as will be discussed later on, the assembly of water channels in a membrane is under the hormonal control of AVP (or ADH). The hormone signals from the activated AVPR through a G protein that causes the elevation of cAMP and the consequent activation of PKA to phosphorylate AQP subunits in the cytoplasm. The phosphorylated subunits migrate to the apical cell membrane, where they form a tetrameric channel , and the process of water uptake is initiated (by itself, the subunit has the property of taking up water but four of them function in a channel). Conversely, the channels can be rendered nonfunctional by dephosphorylation of the subunits. In the kidney glomerulus, about 70% of water in primary urine is reabsorbed by AQP1 and passed into the blood. Precisely 10% more water is reabsorbed by AQP2 at the end of the glomerulus. It is here that ADH/vasopressin acts on AQP2 to increase the reabsorption of water from urine. In humans a deficiency of ADH leads to excessive urination ( diabetes insipidus ), as discussed earlier, in which there can be a urinary output of 10–15 L/day. Conversely, water retention is caused by hypersecretion of ADH , certain drugs, high sodium ion intake, or other conditions. A virtual image of an AQP channel in a cell membrane is shown in Fig. 3.13 .

Fig. 3.14 shows a view from the top of the AQP from red blood cells (AQP1).

In Fig. 3.15 an aquaglyceroporin subunit is shown.

The many AQPs are distributed to different cell types. AQP1 is present mostly in red blood cells , kidneys, and the choroid plexus ( CP ) (the CP is a structure lining the ventricular system in the brain where cerebrospinal fluid is produced). The CP can be an entrance point for some molecules or particles (e.g., viruses) forming a means to bypass the blood–brain barrier . AQP4 is located in the brain, and AQP7 and AQP9 are located in adipocytes . AQP1 and AQP2 are located in the apical and basolateral glomerulus, and AQP2 is in the apical cortical collecting duct of the kidney. AQP3 is in the basolateral outer medullary collecting duct, and AQP4 is located in the basolateral inner medullary collecting duct ( Fig. 3.16 ).

Polycystic kidneys are produced by the disruption of AQP11 following the vacuolization of the proximal tubule. This condition is often inherited and can lead to cysts in other organs and can result in kidney disease, loss of kidney function, and even, ultimately, in death, although there are gradations of this condition. AQP11 and AQP12 are different from the major two classes of AQPs: those that transport water only, and the aquaglyceroporins that transport glycerol, urea, and other small molecules in addition to water. AQP11 is 30 kDa and has been localized to the kidney, testes, liver, and brain. AQP11 is most similar to AQP12 and least similar to AQP4 and AQP7. AQP11 and AQP12 may be the forerunners of a new family of AQPs.