The ATP–ubiquitin–proteasome-dependent proteolytic pathway. 

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... and are involved in Ca + mobilization induced by mechanical stretch [10]. Stretch-activated ion channels were originally identified in chick skeletal myocytes by use of the patch-clamp technique. They are involved in the mechanotransduction of various tissues in ver- tebrates and non-vertebrates [11 ]. Pathophysio- logically a dysfunction of mechanosensitive ion channels have been reported to be involved in stretch- induced arrhythmias [12 ] and in muscular dystrophy [13]. These specialized ion channels are sensitive to changes in membrane tension or stretch which can be induced under experimental conditions by suction applied to the rear of the patch pipette. The activation of the channels shows a graded increase of channel activity with respect to the degree of membrane stretch elicited by different strengths of pipette suction. The electrophysiological properties of the channel, i.e. ion selectivity and channel conductance, remain unaffected by membrane stretch. Endothelial stretch-activated channels have a channel conductance between 25 and 45 pS and are impermeable for anions and permeable for mono- and divalent cations [9,14,15]. The channel permeability for Ca 2 + ions is 3–4 times lower than for monovalent cations e.g. K + and Na + . However, indirect evidence showed that due to the large inwardly directed electrochemical gradient for Ca 2 + ions an activation of the stretch-activated channels leads to a sufficient Ca 2 + influx into the endothelial cell and increases the intracellular Ca 2 + concentration [14,16 ]. Stretch-activated ion channels are blocked by gadolin- ium, a trivalent lanthanide, in concentrations of less than 30 m mol/l. Furthermore, two K + -selective mechanosensitive ion channels have been observed in endothelial cells. An endothelial stretch-activated K + channel (SAC ) has K been identified in freshly isolated rat aortic endothelial cells [17]. This channel had a mean channel conductance in cell-attached patches of 70 pS and was select- ively permeable for K + ions with a permeability ratio K + 5 Na + of 11 5 1. A shear-stress-activated K + channel was observed in cultured bovine aortic endothelial cells [18]. The channel had a mean channel conductance of 31 pS and was inwardly rectifying. The activation of these K + selective mechanosensitive channels led to an efflux of K + ions and subsequently to a hyperpolariz- ation of the endothelium. Correspondingly, endothelial hyperpolarization measured by whole cell current recording or fluorescence dyes has been shown to be elicited by shear stress in bovine aortic and pulmonary endothelial cells [19,20]. Both signals, an increase in intracellular Ca 2 + concentration and cell hyperpolarization are important stimulators of Ca 2 + dependent NO-production in endothelial cells [6,21]. Thus stretch-activated cation channels can act as mechanosensors and transform mechanical stimulation into an intracellular signal leading to a vasodilatory response ( Figure 1). The mechanism by which mechanosensitive forces may control the channel gating process is not well understood. Submembraneous cytoskeletal structures that integrate tension from a wider area of membrane might be involved in channel gating. This was indicated by the observation that disruption of the cytoskeleton by specific drugs alters or abolishes the mechanosensi- tivity of the channels [3 ]. A new type of mechanosensitive ion channels has been identified in the endothelium of intact tissue slices from rat aorta and mesenteric artery [22]. The channel is activated when pressure is applied to the cell membrane via the patch pipette and not by pipette suction. The pressure-activated channel responds to increases of pressure with a graded increase in channel activity. This channel is permeable to monovalent cations and Ca 2 + ions. The Ca 2 + influx through the pressure- activated channel is sufficient to activate neighboring Ca 2 + dependent maxi K + channels. However, also a co-activation of Ca 2 + dependent non-selective cation channels was observed which have a depolarizing effect on the cell potential and could therefore impair endothelial function. Thus, the precise function of the pressure-activated channel remains to be determined by use of specific channel blockers. The channel was almost exclusively observed in intact tissue preparations, rarely in freshly isolated endothelial cells, but not in cultured endothelial cells. This emphasizes the importance of experiments using whole tissue preparations where the target cell is situated in its natural environment. In human and experimental hypertension an endothelial dysfunction has been described. The increased vascular tone in hypertension is in part due to an imbalance in the secretion of vasodilating and vasocon- stricting mediators by the endothelium [23]. Also, the endothelial response to haemodynamic stimulation has been reported to be impaired in hypertension as indicated by a decreased flow-induced endothelium- dependent vasodilatation in spontaneously hypertensive rats compared to normotensive Wistar–Kyoto rats [24]. In adult spontaneously hypertensive rats and in rats with renovascular hypertension (2K1C ) a more than twofold increase of the density of the SAC and K the pressure-activated channel was found [22 ] proving that mechanosensitive channels are regulated in the presence of altered haemodynamic forces. Channel upregulation and associated hyperpolarization by SAC K as well as the increased Ca 2 + influx through the pressure-activated channel would lead to an enhanced vasodilatory response to haemodynamic stimulation. However, it cannot be excluded that coactivation of depolarizing non-selective cation channels by pressure- activated channels, as stated above, would impair endothelial function and contribute to the endothelial dysfunction in hypertension. Endothelium-dependent flow-induced vasodilatation is an important mechanism to protect the vessel wall from damage by increased shear stress associated with increases in blood flow. Mechanosensitive ion channels act as endothelial mechanosensors and mechanotransducers of haemodynamic forces. Upregulation of mechanosensitive ion channels in hypertension might represent an important adaptive mechanism for vessel- wall protection. Amylin, also known as islet amyloid polypeptide, is a 37 amino acid polypeptide co-secreted with insulin by the pancreatic beta cell [1]. Previous research has primarily evaluated the role of this peptide in carbohydrate metabolism with studies concentrating on its role in beta cell function and in the genesis of insulin resistance [1 ]. Amylin has also been reported to have effects on bone metabolism [2], which may ultimately be relevant in renal osteodystrophy, although an exploration of this aspect has not been reported. However, this report will focus on recent findings by our group which indicate that amylin acts in the kidney probably via a specific G-protein coupled receptor [3]. These effects of amylin have potential implications for various pathological conditions including hypertension and the renal response to injury. These findings are of particular interest since the amylin analogue, pramlin- tide, is now in phase III clinical trials as an agent to control postprandial hyperglycaemia in diabetes [4]. High-affinity amylin binding sites have been detected in both the kidney [3 ] and certain regions of the central nervous system [5 ]. Using in vitro autoradiographic techniques, we have described high-affinity sites of amylin binding ( Kd ~ 1 nM ) in the rat renal cortex [3 ]. The pharmacological characteristics of these binding sites, as determined by the pattern of inhibition using peptide antagonists, was very similar to those previously described in the rat brain [5,6 ]. Amylin is a member of the same homologous group of peptides as calcitonin, adrenomedullin and calcitonin gene- related peptide [1]. Thus far, the calcitonin (CTR), ...

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... was implanted. Because patients with fixed hyperphosphataemia might elaborate a similar renal phosphate transport inhibitor, we sought the presence of such a factor in dialysates obtained from patients dialysed with polysul- phone membranes, which have a 30 000–40 000, Dalton cutoff, and would allow such a molecule to pass through [4]. We have characterized such a factor in these dialysates. It appears to differ from that in tumour cells in that it is of somewhat larger molecular weight and inhibits glucose and alanine transport in addition to inhibiting phosphate transport. Information from the Hyp mouse and from patients with X-linked hypophosphataemic rickets suggests that tumour-induced osteomalacia and the former conditions may be related [5,6 ]. Meyer et al. showed with parabiosis experiments that Hyp mice with hypophosphataemia and renal phosphate wasting, produce a substance that causes hypophosphataemia in the normal mouse of a normal– Hyp parabiotic pair [5,6,]. Reversing the parabiosis normalizes serum phosphorus in the normal mouse. The factor is not PTH, as parathyroidectomizing Hyp mice does not alter their ability to produce hypophosphataemia in normal animals of a parabiotic pair. Nesbitt and others showed that a normal kidney transplanted into a Hyp mouse, exhibits a reduced tubular maximum for phosphate [6 ]. Conversely, a Hyp mouse kidney transplanted in normal mouse ceases to waste phosphate in the urine. Recently a mutant gene (PEX ) was isolated by the Hyp Consortium from patients with X-linked hypophosphataemic rickets [9 ]. The abnormal protein was not a phosphate transporter but was related to a family of endopeptidases. It is very likely that the product encoded by this gene does not directly influence phosphate transport. It may, however, break down a normal circulating factor that causes hypophosphataemia. Thus the mutated gene might be responsible for the hypophosphataemia seen in these patients by allowing an excess of this hypophosphataemic factor to circulate. In conclusion, there is persuasive evidence that a new factor, ‘phosphatonin’, may be responsible for the alteration in phosphate transport seen in various diseases such as tumour-induced osteomalacia, X-linked hypophosphataemia, and hypophosphataemia in the Hyp mouse and the gyroscopic mouse. ‘Phosphatonin’ may be abnormally low in patients with tumoural calcinosis, an unusual disease associated with elevated serum phosphorus, and 1,25-dihydroxyvitamin D concentrations, and ectopic deposition of calcium and phosphorus. ‘Phosphatonin’ may be appropriately elevated in patients with renal failure who have phosphate retention. The study of inhibitors of renal sodium phosphate transport and the characterization of these factors will yield insights into the control of phosphate transport and balance. The turnover of protein in normal adults is enormous, averaging 280 g/day [1], about 90% being intracellular as mammalian cells continually degrade and replace protein. The degradative rates of cellular proteins vary widely: some cytosolic enzymes have half-lives as short as 20 min while others last for days, and the majority of rat hepatocyte proteins are replaced in days but in muscle or brain, the process takes weeks. Clearly, the cell’s proteolytic mechanisms must be highly selective and tightly regulated. If this were not true, uncontrolled destruction of essential proteins or failure to degrade short-lived regulatory proteins would drastically alter cell function. Dramatic increases in our understanding of the mechanisms and regulation of protein degradation have implications for treating human diseases. Controlled destruction of cell proteins serves important homeostatic functions [1 ]: (1 ) rapid removal of rate-limiting enzymes and regulatory proteins is critical in controlling growth and metabolism (e.g. the pro- grammed destruction of cyclins, the cell cycle regulatory proteins); (2 ) proteolysis allows cells to adapt to physiological conditions (e.g. during fasting, hepatic enzymes for glucose storage disappear while gluconeogenic enzymes accumulate, but within hours of refeeding this pattern reverses); (3 ) selective elimination of damaged or improperly folded proteins (e.g. after oxygen radical- damage or the mutated transmembrane regulator protein in cystic fibrosis; (4 ) proteolysis is required by the immune system in its surveillance for cancer or virus- infected cells [2 ]; (5) an inadequate diet or catabolic diseases stimulate the degradation of cell proteins to provide amino acids for gluconeogenesis and protein synthesis. In all these cases, the ATP–ubiquitin–proteasome pathway plays a critical role. The ATP–ubiquitin–proteasome pathway degrades proteins by a novel mechanism and functions in the nucleus and cytoplasm of all cells. Proteins degraded by this pathway are first ‘marked’ by covalent conjugation to the small protein cofactor, ubiquitin, in a multistep process requiring three enzymes and ATP (Figure 1 ) [3 ]. After a chain of ubiquitin molecules is added, the protein can be recognized by the large 26S proteasome complex in another ATP-dependent process which releases ubiquitin for recycling. The proteolytic core of this complex is the 20S particle consisting of two inner rings, each consisting of 7 beta-type subunits positioned between two outer rings, each consisting of 7 alpha-type subunits [3]. After another ATP-dependent reaction, which releases ubiquitin for recycling, the protein is unfolded and degraded by a novel proteolytic mechanism utilizing threonine at the amino terminus of each beta-type subunit to catalyse cleavage of peptide bonds. This mechanism digests the entire protein to small peptides (i.e. a processive mechanism) which are rapidly degraded to amino acids by cytoplasmic peptidases [3]. How is the cell protected from the disaster of non- specific proteolysis by this pathway? Selectivity depends on the ubiquitin conjugation process because cells contain a variety of ubiquitination enzymes that are specific for different types of proteins (e.g. specific E2 and E3 enzymes combine to catalyse ubiquitination of the individual cyclins to regulate mitosis) [1,3]. Second, the active sites of the 26S proteasome are isolated within its central chamber and only unfolded proteins can enter the narrow opening at the ends of the proteasome complex; unfolding requires recogni- tion of the ubiquitin chain. These characteristics and ATP consumption at multiple steps provide a remark- able degree of selectivity, efficiency, and control of protein degradation. In catabolic illnesses, the loss of muscle results largely from accelerated breakdown of the long-lived, myofibrillar proteins (actin and myosin) which comprise 60–70% of muscle protein. In response to acidosis, infection, or certain tumours, skeletal muscle protein is lost preferentially while visceral organs (e.g. the kidney and liver) lose little or no protein, and the brain is unaffected. Studies of rat muscles during fasting, acidosis, or denervation atrophy demonstrated that the loss of muscle is primarily due to activation of the ubiquitin–proteasome pathway [4,5]. When atrophying muscles were incubated in vitro with agents that blocked the activity of lysosomal or calcium-activated proteases (calpains), the accelerated proteolysis per- sisted. However, inhibitors of ATP production reduced muscle protein degradation to levels in control muscles. Increased levels of ubiquitin-conjugated proteins in muscles coincident with maximal protein degradation provided more definitive evidence for activation of the ubiquitin-proteasome pathway [6 ]. There also were increased levels of the mRNAs encoding ubiquitin and subunits of the proteasome during fasting, acidosis, and denervation atrophy despite a lower RNA content of muscles [4]. In rats with acidosis from acute or chronic uraemia (CRF ), there is increased muscle protein degradation and mRNAs encoding components of the ubiquitin–proteasome pathway [1,7]. In CRF, correcting acidosis blocks the accelerated proteolysis and prevents the rise in mRNAs of ubiquitin and proteasome subunits [7]. These findings point to coord- inated adaptations that enhance the proteolytic capacity of the ubiquitin-proteasome pathway and favour muscle wasting. In experimental models that mimic muscle-wasting conditions, there is consistent activation of the ATP- dependent proteolytic pathway causing muscle protein breakdown [1 ]. Presumably this response evolved to provide the injured or infected organism with amino acids for energy and synthesis of new proteins (e.g. acute-phase reactant proteins) but if prolonged, muscle wasting occurs. For example, within hours of injecting endotoxin, or live bacteria, or puncture of the caecum, the degradation of myofibrillar proteins by an ATP- dependent process rises sharply [1,8]. After thermal injury, protein catabolism increases markedly in muscles distant from the injury and ATP-dependent proteolysis rises concurrently with ubiquitin mRNA [9 ]. The ubiquitin–proteasome pathway appears to be responsible for muscle wasting in trauma patients, since mRNAs encoding components of the pathway are high in muscle biopsies [10 ]. Muscle wasting in cancer cannot be explained simply by anorexia, since tumour-bearing rats show greater muscle loss and higher rates of proteolysis compared to rats fed ident- ical amounts of food [11]. Again, cancer stimulates activity of the ATP-dependent proteolytic pathway and increases ubiquitin mRNA in muscle. The increased levels of mRNAs encoding ubiquitin and proteasome subunits in catabolic states suggest activation of a common genetic programme to enhance the expression of pathway components, and hence the capacity of this degradative pathway. Recently nuclei were isolated from muscles of CRF or insulinopenic rats and it was found that both conditions stimulate transcription of genes for polyubiquitin and proteasome subunits despite a ...