Derangements in systemic and local metabolism develop in patients with CHF [chronic HF (heart failure)] and contribute to the progression of the disease. Impaired skeletal muscle metabolism, morphology and function leading to exercise intolerance are hallmarks of the syndrome of CHF. These changes result in abnormal glucose and lipid metabolism, and the associated insulin resistance, which contribute to progression of skeletal muscle catabolism and development of muscle atrophy in patients with advanced HF. In the present issue of Clinical Science , Toth and co-workers demonstrate the impairment of skeletal muscle protein metabolism in patients with HF, and specifically show an impaired anabolic response in the skeletal muscle of these patients following a period of nutritional deficiency.

Patients with chronic HF (heart failure) experience muscle atrophy during the course of the disease. The mechanisms underlying muscle atrophy in HF, however, are not understood. Thus we evaluated leg phenylalanine balance and kinetics in HF patients and controls following a brief fast (24 h) and under euglycaemic–hyperinsulinaemic–hyperaminoacidaemic conditions to determine whether HF increases muscle protein catabolism in response to nutritional deprivation and/or diminishes the anabolic response to meal-related stimuli (insulin and amino acids) and whether alterations in protein metabolism correlate to circulating cytokine levels. No differences in phenylalanine balance, rate of appearance or rate of disappearance were found between patients and controls under fasting conditions. However, the anabolic response to hyperinsulinaemia–hyperaminoacidaemia was reduced by more than 50% in patients compared with controls. The diminished anabolic response was due to reduced suppression of the leg phenylalanine appearance rate, an index of protein breakdown, in HF patients; whereas no group difference was found in the increase in the leg phenylalanine disappearance rate, an index of protein synthesis. The diminished responses of both phenylalanine balance and appearance rate to hyperinsulinaemia–hyperaminoacidaemia were related to greater circulating IL-6 (interleukin-6) levels. Our results suggest that, following a brief period of nutritional deprivation, HF patients demonstrate an impaired muscle protein anabolic response to meal-related stimuli, due to an inability to suppress muscle proteolysis, and that this diminished protein anabolic response correlates with markers of immune activation. The inability to stimulate muscle protein anabolism following periods of nutritional deficiency may contribute to muscle wasting in HF patients.

Pepsin, acid and Helicobacter pylori are major factors in the pathophysiology of peptic ulcer disease and reflux oesophagitis. Ecabet sodium reduces the survival of H. pylori in the stomach and inhibits pepsin activity in the gastric juice of experimental animals. Here we have investigated the effects of ecabet sodium on some of the factors involved in the dynamics of the mucosal barrier, i.e. pepsins and mucins. This study used gastric juice obtained from 12 non-symptomatic volunteers and nine patients with reflux oesophagitis. Ecabet sodium significantly inhibited pepsin activity in human gastric juice, with a maximum inhibition of 78%. Pepsin 1, the ulcer-associated pepsin, was inhibited to the greatest extent. The ability of gastric juice to digest mucin was significantly inhibited by ecabet. As with gastric juice proteolytic activity, the inhibitory effect of ecabet on mucolysis was greater in gastric juice from patients with reflux oesophagitis than in that from controls. Ecabet sodium showed a positive interaction with gastric mucin, as assessed by an increase in viscosity. Thus ecabet sodium may reduce the aggressive potential of gastric juice towards the mucosa, which may be relevant in the treatment of reflux oesophagitis and peptic ulcer disease. In addition, it may strengthen the mucus barrier in peptic ulcer disease and gastritis.

There is evidence that burn injury stimulates ubiquitin–proteasome-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, protein substrates are conjugated to multiple molecules of ubiquitin, whereafter they are recognized, unfolded and degraded by the multicatalytic 26 S protease complex. The 20 S proteasome is the catalytic core of the 26 S protease complex. The influence of burn injury on the expression and activity of the 20 S proteasome has not been reported. We tested the hypothesis that burn injury increases 20 S proteasome activity and the expression of mRNA for the 20 S proteasome subunits RC3 and RC7. Proteolytic activity of isolated 20 S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from burned rats. Northern-blot analysis revealed that the expression of mRNA for RC3 and RC7 was increased by 100% and 80% respectively following burn injury. Increased activity and expression of the 20 S proteasome in muscles from burned rats support the concept that burn-induced muscle cachexia is at least, in part, regulated by the ubiquitin–proteasome proteolytic pathway.

1. Burn injury stimulates ubiquitin-dependent protein breakdown in skeletal muscle. The 20S proteasome is the proteolytic core of the 26S proteasome that degrades ubiquitin conjugates. We examined the effects of the proteasome inhibitors N -acetyl- l -leucinyl- L -leucinal- l -norleucinal (LLnL), lactacystin and β-lactone on protein breakdown in muscles from burned rats. 2. A full-thickness burn of 30% total body surface area was inflicted on the back of rats. Control rats underwent a sham procedure. After 24 ;h, extensor digitorum longus muscles were incubated in the absence or presence of 20S proteasome blocker and protein turnover rates and ubiquitin mRNA levels were determined. 3. LLnL resulted in a dose- and time-dependent inhibition of total protein breakdown in incubated muscles from burned rats. Lactacystin and β-lactone blocked both total and myofibrillar muscle protein breakdown. In addition to inhibiting protein breakdown, LLnL increased ubiquitin mRNA levels, possibly reflecting inhibited proteasome-associated RNase activity. 4. Inhibited muscle protein breakdown caused by LLnL, lactacystin and β-lactone supports the concept that the ubiquitin–proteasome pathway plays a central role in burn-induced muscle proteolysis. Because the proteasome has multiple important functions in the cell, in addition to regulating general protein breakdown, further studies are needed to test the role of proteasome blockers in the treatment or prevention of muscle catabolism.

1. We report here the extent to which changes in protein turnover contribute to the previously described inhibition of growth of rat tibial length and skeletal muscle mass in response to protein deficiency [1], energy restriction and corticosterone treatment [2]. Measurements of 35 S uptake in vivo also enabled the qualitative pattern of changes in proteoglycan synthesis in bone and muscle to be established. 2. Protein deficiency was examined by ad libitum feeding of 20%, 7%, 3.5% and 0.5% protein diets with measurements at 1, 3 and 7 days (all diets), and 14 and 21 days (0.5% protein). In bone this induced delayed inhibition of tibial growth with parallel inhibition of protein synthesis, as measured by the phenylalanine flooding dose method. This was mediated by reductions in both ribosomal capacity (RNA/protein ratio) and activity (protein synthesis/RNA) in the 0.5% protein group. The pattern of inhibition of proteoglycan sulphation, measured as 35 S uptake 60 min after injection of a tracer dose of labelled sulphate, was similar to that of protein synthesis. 3. In muscle there was an intermediate graded inhibition of protein synthesis by protein deficiency, mediated by reductions in both ribosomal capacity and activity in the 0.5% protein group, which preceded growth inhibition in the 7% and 3.5% groups, and which was progressive with time. Transient increases in proteolysis contributed to the growth inhibition is some groups, but the rate fell eventually in the 0.5% group. The pattern of response of proteoglycan sulphation differed from protein synthesis with a delayed inhibition, but with subsequent marked reduction. 4. Energy restriction was induced by diets fed for 4 or 8 days at 75%, 50% and 25% ad libitum intakes with protein intakes held constant, and corticosterone treatment involved a dose of 10 mg day −1 100 −1 g (subcutaneous) with ad libitum feeding. In bone this induced a pattern of length growth inhibition which was dissociated from inhibition of protein synthesis in the moderately restricted (75% and 50%) groups. Only in the 25% group and in the 8 day corticosterone group was protein synthesis inhibited, through reductions in ribosomal capacity and activity. 35 S uptake was also dissociated from growth inhibition, with reduced 35 S uptake observed only after corticosterone treatment or 8 days of the 50% or 25% diets. 5. In muscle the energy restriction and corticosterone treatment induced parallel inhibitions of growth and protein synthesis, mediated by similar graded reductions in the RNA/protein ratios and in the 25% group in the K RNA . Proteolysis was unchanged in all except the 4-day corticosterone group (elevated by 25%) and the day 8 25% group (elevated by 40%) and corticosterone group (elevated by 60%). 35 S uptake was inhibited in parallel to muscle growth and protein synthesis. 6. These data show that inhibition of protein synthesis and 35 S uptake is an invariable element of muscle growth inhibition, and a usual but not invariable element of bone growth inhibition. Partial correlation analysis of the interactions between dietary protein, bone growth and muscle protein and proteoglycan synthesis shows that bone growth (as indicated by epiphyseal cartilage width) is significantly correlated with muscle protein synthesis and especially 35 S uptake, suggesting that the regulation of muscle growth by passive stretch consequent on bone lengthening includes muscle connective tissue growth as an important target.

1. Using the forearm balance method, together with systemic infusions of l-[ ring -2,6- 3 H]phenylalanine and l-[1- 14 C]leucine, we examined the effects of infused branched-chain amino acids on whole-body and skeletal muscle amino acid kinetics in 10 postabsorptive normal subjects; 10 control subjects received only saline. 2. Infusion of branched-chain amino acids caused a four-fold rise in arterial branched-chain amino acid levels and a two-fold rise in branched-chain keto acids; significant declines were observed in circulating levels of most other amino acids, including phenylalanine, which fell by 34%. Plasma insulin levels were unchanged from basal levels (8 ± 1 μ-units/ml). 3. Whole-body phenylalanine flux, an index of proteolysis, was significantly suppressed by branched-chain amino acid infusion ( P < 0.002), and forearm phenylalanine production was also inhibited ( P < 0.03). With branched-chain amino acid infusion total leucine flux rose, with marked increments in both oxidative and non-oxidative leucine disposal ( P < 0.001). Proteolysis, as measured by endogenous leucine production, showed a modest 12% decrease, although this was not significant when compared with saline controls. The net forearm balance of leucine and other branched-chain amino acids changed from a basal net output to a marked net uptake ( P < 0.001) during branched-chain amino acid infusion, with significant stimulation of local leucine disposal. Despite the rise in whole-body non-oxidative leucine disposal, and in forearm leucine uptake and disposal, forearm phenylalanine disposal, an index of muscle protein synthesis, was not stimulated by infusion of branched-chain amino acids. 4. The results suggest that in normal man branched-chain amino acid infusion suppresses skeletal muscle proteolysis independently of any rise of plasma insulin. Muscle branched-chain amino acid uptake rose dramatically in the absence of any apparent increase in muscle protein synthesis, as measured by phenylalanine disposal, or in branched-chain keto acid release. Thus, an increase in muscle branched-chain amino acid concentrations and/ or local branched-chain amino acid oxidation must account for the increased disposal of branched-chain amino acids.

1. Mucolytic (mucus solubilizing) activity in human faeces has been characterized with both purified human and pig colonic mucin and shown to be mediated by proteolysis. 2. Mucolytic activity was demonstrated by: (i) a drop in mucin viscosity; (ii) a substantial reduction in mucin size, from polymer to degraded subunit, as assessed by Sepharose CL-2B gel filtration; (iii) formation of new N -terminal peptides. 3. Mucolytic activity was also followed in faecal extracts by its proteolytic activity using standard succinyl albumin substrate. Proteolysis extended over the pH range 4.5–11.0. Proteolysis was inhibited at pH 7.5 by soybean trypsin inhibitor and phenylmethanesulphonyl fluoride, suggesting the presence of serine proteinases. 4. The polyacrylate carbomer (934P) inhibited both mucolysis of pig colonic mucin and proteolysis of succinyl albumin. 5. Interaction between the polyacrylate (carbomer 934P) and purified human and pig colonic mucin was demonstrated by a marked synergistic increase in solution viscosity (360% above control). 6. The results demonstrate the presence of a mucolytic activity in the human colonic lumen that has the potential to degrade the mucus barrier, and that polyacrylates inhibit this mucolysis and interact to strengthen the colonic mucus barrier. Polyacrylates may therefore have therapeutic potential in inflammatory bowel disease where luminal proteolytic activity can be raised.

1. Neutrophils from patients with chronic obstructive bronchitis and emphysema or age-matched control subjects were cultured on a substrate of 125 I-fibronectin. The neutrophils from patients with lung disease digested significantly more fibronectin and released more elastase into the culture supernatant than did cells from control subjects. Preincubation of neutrophils from emphysematous patients with plasma from control subjects significantly inhibited fibronectin digestion by the patients' neutrophils by, on average, 10%. Preincubation of control subjects' neutrophils with plasma from emphysematous patients had no effect on fibronectin digestion. 2. Tumour necrosis factor increased fibronectin digestion in a dose-dependent manner when the cytokine was added to the adherent cells but not when preincubated with the polymorphonuclear leucocytes in suspension. Bacterial endotoxin in concentrations above 6 μg/ml significantly increased fibronectin digestion by neutrophils, but leukotriene B 4 , interferon-μ and interleukin-1α had no significant effects. 3. Dexamethasone inhibited fibronectin digestion by neutrophils in a dose-dependent manner, from 11% at 10 −10 mol/l to 68% at 10 −3 mol/l.

1. The quantification of α 1 -antitrypsin (α 1 AT) by standard immunological techniques is altered by interaction of the protein with leucocyte elastase. 2. The results obtained for α 1 -antitrypsinleucocyte elastase mixtures in the presence of a functional excess of the inhibitor were relatively accurate for the first 6 h. However, continued incubation for more than 24 h led to a major over-estimation of the α 1 AT as the result of breakdown of the enzyme-inhibitor complex releasing a partially proteolysed form of the inhibitor. 3. In the presence of excess enzyme up to a twofold overestimation of α 1 AT occurred within 1 h and the degree of overestimation increased with time (up to threefold at 24 h). This was eventually associated with the presence of only a partially proteolysed form of α 1 AT (mol wt. ⋍ 50000). 4. Different results for each sample were obtained when different polyclonal antisera were used to quantify the α 1 AT. 5. Complete inactivation of α 1 AT by oxidation resulted in little change in the immunological quantification of the protein. However, further addition of H 2 O 2 led to a progressive underestimation of the α 1 AT. 6. The effect of physicochemical alteration on the immunological quantification of α 1 AT by different antisera should be borne in mind for all studies assessing this protein in lung secretions.

The pathogenesis of many acute and chronic lung diseases remains a mystery. However, recent years have seen a rapidly increasing interest in the role of proteolytic enzymes and their inhibitors in modifying the inflammatory, destructive and reparative changes that occur in the lung. Much of this interest owes its existence to two observations in the early 1960s: firstly, the recognition that subjects with an inherited deficiency of α 1 -antitrypsin (α 1 -AT; the main serum inhibitor of proteolytic enzymes) had a high incidence of pulmonary emphysema [1], and secondly the demonstration by Gross et al. [2] that a proteolytic enzyme (papain) was capable of producing lesions similar to emphysema in experimental animals. These observations ultimately led to the proteinase—anti-proteinase theory of emphysema, which predicts that a state of balance occurs in the healthy lung in which the proteolytic enzyme inhibitors functionally equal or exceed the enzymes. Destructive lung disease occurs when the enzymes functionally exceed the inhibitors such that they remain active within the lung, resulting in digestion of connective tissue. This general concept of a disturbed proteinase—anti-proteinase balance within the lung has been recently applied to many other lung diseases, and some will be mentioned later. However, it is in the study of chronic bronchitis and emphysema that the concept has become most well established.