Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data.

OBJECTIVES: Heregulins (HRGs) are known to induce expression of angiogenic factors such as cysteine rich-61 (CYR61) and collectively to promote neoangiogenesis. Along with extracellular matrix remodelling, mediated by matrix metalloproteinases (MMPs), these factors are important in atherogenesis. The aim of the present study was to investigate HRG, CYR61 and MMP-9 expression and their relationship with clinical and histopathological findings in carotid occlusive disease. MATERIALS AND METHODS: Specimens of human carotid atherosclerotic plaque (n=90) were obtained by endarterectomy. Expression of HRG, CYR61 and MMP-9 was assessed by immunohistochemical and Western blot analysis. Associations between protein expression and degree of carotid stenosis, presence of symptoms, presence of an infarct in CT scan and carotid plaque histopathology were investigated. RESULTS: An increase in HRG, CYR61 and MMP-9 expression was found, particularly in neovascularized regions of the plaques. High HRG expression was associated with the degree of carotid stenosis (p=0.028) and plaque histopathology (p=0.002). More than half of specimens from plaques with >90% stenosis had intense expression of CYR61 (p=0.047). Increased expression of MMP-9 was associated with degree of stenosis and presence of cerebral infarct on CT scan (p=0.05). CONCLUSION: HRG, CYR61 and MMP-9 are highly expressed in human atherosclerotic carotid plaques. The association with the degree of stenosis and/or plaque histopathology implies an involvement in lesion progression.

Role of cyclic AMP and inorganic phosphate in the regulation of muscle glycogenolysis during exercise.

The roles of cAMP and inorganic phosphate (Pi) in the regulation of muscle glycogenolysis during exercise have been investigated in humans using the needle biopsy technique. The fraction of phosphorylase a in resting muscle was as a mean 23%, but the rate of glycogenolysis was extremely low. Epinephrine infusion increased cAMP in muscle by 3-fold and transformed 80% of phosphorylase to the a form. Despite this, the rate of glycogenolysis was only 5-10% of the maximum rate of phosphorylase a (Vmax a) determined in vitro. Isometric exercise for 25 s at 66% MVC or electrical stimulation for 50 s at 20 Hz transformed about 53% and 80% of phosphorylase in the a form. The rate of glycogenolysis ranged between 50-90 mmol.kg-1.dm.min-1 and was close to Vmax of phosphorylase a determined in vitro. No significant difference in the rate of glycogenolysis in muscle was observed after isometric exercise to fatigue without and with epinephrine infusion, respectively. Apparently the rate of glycogenolysis in muscle is not solely related to the fraction of phosphorylase in the a form. Several factors could be responsible for allosteric and/or substrate regulation. The results in the present studies can be explained on the basis of substrate regulation of phosphorylase activity, provided that Pi is present in a limiting amount at the active site of phosphorylase in muscle at rest. It is concluded that transformation of phosphorylase b to a is important but alone is not adequate for a high activity and thus for a high rate of glycogenolysis in muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle glucolysis, glycogenolysis and glycogen phosphorylase during electrical stimulation in man.

Phosphorylase activity, glycogenolytic and glucolytic rates were estimated in human quadriceps muscle during electrical stimulation at 20 Hz. Two stimulation periods of 10 s duration were separated by a pause of 60 s. The blood circulation to the leg was intact or occluded during the experiment. ATP turnover rates and force production were of the same order during the two contraction periods both with and without intact blood flow. Also the increase in phosphorylase a activity (from approximately 30% to approximately 65%) was the same during the contraction periods. Glycogenolytic and glucolytic rates were however about 30% higher (P less than 0.05) during the second contraction compared with the first when circulation was occluded, but similar when the circulation was intact. During the 60 s rest period, the phosphocreatine (PCr) was maintained at a low level and inorganic phosphate (Pi) remained increased under occluded circulation while PCr was resynthesized in the rest period with intact circulation. We conclude that the increased glycogenolytic rate observed during the second contraction with occluded blood circulation was due to the high [Pi] in the muscle and that the increased glucolytic rate was caused by high [Pi] and low [PCr]. In the rest period with anoxia the glycogenolysis was completely inhibited and glucolysis was inhibited by 95% in spite of the changes in [PCr] and [Pi].

ATP utilization and force during intermittent and continuous muscle contractions.

Energy utilization and force generation under anaerobic conditions were studied in electrically stimulated quadriceps femoris muscle of four volunteers. To investigate the effects of intermittent vs. continuous stimulation one leg was stimulated intermittently and the other continuously during 50 s. The same initial force was produced, and biopsy samples were obtained before the stimulation and after 10, 20, and 50 s and analyzed for energy-rich phosphagens, glycolytic intermediates, and phosphorylase. The ATP utilization and glycolysis were greater during intermittent contraction, but glycogenolysis was equal. ATP content decreased to lower values after intermittent contraction (16.4 compared with 19.6 mmol/kg dry muscle after continuous contraction). Force generation was well preserved during continuous contraction but successively decreased after 20 s of intermittent stimulation down to 50% of initial at end of work. The energy cost per unit work was greater during intermittent stimulation and increased with contraction time, whereas it decreased with time during continuous stimulation. The decrease in force generation in intermittent exercise is suggested to be due to the higher energy cost for contraction resulting in greater changes in the intracellular environment with lower ATP and increased H+ and Pi. These changes would decrease both activation of the contractile system and the cross-bridge turnover rate resulting from activation.

Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles.

This study investigated the effect of adrenaline infusion on the cAMP content, glycogen phosphorylase activity and the rate of glycogen breakdown in rat extensor digitorum longus (EDL) and soleus muscles. Adrenaline was constantly infused in a dose of 0.15 micrograms kg-1 body wt min-1. The cAMP content increased approximately 2.8-fold in both muscles after 2 min of infusion. Phosphorylase a + b activity was six times higher in fast-twitch muscle (EDL) than in slow-twitch (soleus) and remained unchanged during the infusion. Phosphorylase a activity increased by 8.4-fold in EDL and 2.4-fold in soleus muscles during the infusion period. Glycogen content decreased in EDL muscle by 10% whereas no change was observed in soleus. It is concluded that beta-adrenergic stimulation by adrenaline results in a similar cAMP increase in both muscles. The low rate of glycogen breakdown in EDL and the unchanged content of glycogen in soleus muscle suggest that cAMP mediated transformation of phosphorylase b to a in itself is not adequate for a rapid glycogenolysis in muscle.

The effect of adrenaline infusion on the regulation of glycogenolysis in human muscle during isometric contraction.

The regulation of glycogenolysis in human muscle during isometric contraction without and with adrenaline infusion has been investigated. The content of cAMP in muscle increased three-fold during the infusion. Total glycogen phosphorylase and synthetase activities were unchanged during contraction without and with adrenaline infusion. The fraction of phosphorylase in the a form was in resting muscle 26% and at the end of contraction 24%. During adrenaline infusion phosphorylase a increased to 80%. Contraction during continued infusion resulted in a decrease of phosphorylase a to 42%, despite persistently increased cAMP content in muscle. The activity of synthetase I decreased to about half of the initial value during adrenaline infusion and contraction both without and with the infusion. The rate of glycogenolysis in muscle during contraction was not significantly changed by the infusion. Phosphocreatine (PCr) decreased during the contraction and the decrease was similar without and with adrenaline infusion. The amount of inorganic phosphate (Pi) accumulated in muscle during contraction was lower when adrenaline was given due to a greater accumulation of hexose-monophosphates. It is concluded that the rate of glycogenolysis in muscle during contraction without and with adrenaline infusion is a function both of phosphorylase in the form a form and the availability of Pi at the active site of the enzyme.

Activation of glycogen phosphorylase by electrical stimulation of isolated fast-twitch and slow-twitch muscles from rat.

The influence of muscle contraction, induced by electrical stimulation, on the activity of glycogen phosphorylase, the contents of high-energy phosphates, hexose-monophosphates and lactate have been studied in isolated extensor digitorum longus (EDL) and soleus muscles from rats. The activity of phosphorylase a + b was about nine times higher in fast twitch muscles (EDL) than in slow-twitch soleus and remained unchanged during the stimulation. A pronounced increase of phosphorylase a occurred during the stimulation in EDL muscle. Stimulation with a frequency of 50 Hz for 10 s and 2 Hz for 90 s resulted in a 44-fold and five-fold increase in phosphorylase a, respectively. In contrast, stimulation of soleus muscle resulted in only a minor increase of phosphorylase a. The rate of glycogenolysis increased in both muscles during the stimulation but the increase was four to five times higher in the EDL than in soleus muscle. The content of phosphocreatine (PCr) before stimulation was much higher in EDL than in soleus but similar after the stimulation. This resulted in a three- to four-fold higher release of inorganic phosphate (Pi) in EDL than in soleus during contraction. Pi has previously been shown to be present in a limiting amount for the activity of phosphorylase and the increase during contraction is of importance for increasing the glycogenolytic rate. It is concluded that the higher glycogenolytic capacity in fast-twitch muscles compared to slow-twitch muscles is due to: (1) higher content of phosphorylase a + b, (2) higher degree of transformation of the enzyme into the a form during contraction, and (3) higher content of PCr, which liberates a large amount of Pi during contraction.

The effect of circulatory occlusion on the glycogen phosphorylase-synthetase system in human skeletal muscle.

The effect of circulatory occlusion upon the glycogen phosphorylase-synthetase system in intact human muscle at rest has been investigated using the needle biopsy technique. The fraction of phosphorylase in the a form was 26% before occlusion and decreased to 9% after 40 min of occlusion. Synthetase I activity was unchanged during occlusion. After 40 min of occlusion the content of phosphocreatine was decreased by 40%, with a corresponding increase in creatine and inorganic phosphate (Pi). The observed glycogenolytic rate increased during occlusion up to 0.8 mmol glycosyl units kg-1 dry muscle min-1. An intracellular Pi concentration at rest of 2.0 mmol l-1 was calculated from the activities of phosphorylase a and synthetase I assuming that under these conditions they are equal. It is concluded that the glycogenolytic rate during occlusion is a function of both the fraction of phosphorylase in the a form and the availability of Pi at the active site of the enzyme.

Effects of beta-blockade on glycogen metabolism in human subjects during exercise.

The effect of beta-blockade on glycogen metabolism during isometric and dynamic exercise in humans has been investigated. Isometric exercise increased the glycogenolytic rate in muscle but had no effect on the cAMP content. Neither the metabolic pattern nor the time of contraction was affected by beta-blockade. Dynamic exercise increased the cAMP content in muscle by about 100%. The cAMP content at rest was significantly reduced after propranolol infusion and did not increase during exercise. Total hexosemonophosphates increased sixfold during exercise but little or no increase occurred after administration of propranolol. The accumulation of lactate in muscle was slightly reduced during exercise following beta-blockade. The fraction of phosphorylase in the alpha form was 22.5% of the total at rest but decreased to 16% at exhaustion. Synthetase I was similarly decreased. During exercise with propranolol phosphorylase alpha decreased further to 3%, whereas synthetase I was unchanged. It is concluded that beta-blockade has no effect on muscle glycogenolysis during isometric contraction but decreases the rate of glycogen degradation during dynamic exercise at high work loads due to changes in the phosphorylase-synthetase system.

Acidotic depression of cyclic AMP accumulation and phosphorylase b to a transformation in skeletal muscle of man.

Intravenous infusion of adrenaline was performed in three healthy subjects on two occasions. In one case subjects performed a maximal isometric contraction before infusion. Biopsies were taken from the quadriceps femoris muscle before and after infusion for 0.5 and 2 min, and analysed for muscle pH, cyclic AMP, metabolites and activities of glycogen phosphorylase and synthetase. Isometric contraction resulted in a decrease of muscle pH to 6.60 (normal value at rest 7.0-7.1). By this experimental procedure the effect of adrenaline infusion could be studied on a muscle with normal pH and one with low pH. Cyclic AMP increased from 3 to about 9.5 mumol per kg dry weight after 0.5 min of adrenaline infusion. When isometric contraction preceded the infusion, cyclic AMP increased more slowly and was about 5.5 mumol per kg dry weight after the same time of infusion. Phosphorylase a constituted about 22% of total phosphorylase in resting muscle but increased rapidly to 80% after 0.5 min infusion. When exercise preceded infusion phosphorylase a decreased and was still lower after 2 min infusion. The results can be explained by inhibition of adenylcyclase and phosphorylase b kinase at low muscle pH.

The cyclic-AMP concentration in plasma and in muscle in response to exercise and beta-blockade in man.

At rest the cAMP concentration in (muscle samples of) the quadriceps femoris ranged from 1.55 to 3.00 mumol per kg dry muscle and in plasma from 15.3 to 32.3 nmol per 1. Blockade of the beta adrenoreceptors with propranolol resulted in a significant decrease in the concentration in muscle at rest, the magnitude of the fall being related to the initial level. Similarly in plasma there was a trend towards lower levels of cAMP in those with the highest pretreatment levels, but the overall change was not statistically significant. There was no relation between the concentrations in muscle and plasma, before or after beta-blockade. Maximum dynamic exercise for 4-8 min resulted in an approximate doubling in the cAMP concentration in both muscle and blood. The increase in plasma was closely related to that in muscle. Beta-blockade inhibited totally the rise in cAMP in muscle during exercise but was marginally less effective in preventing the increase in blood. No increase in plasma or muscle cAMP levels during 40-70 s isometric contraction were observed.

Regulation of glycogenolysis in human muscle in response to epinephrine infusion.

The regulation of glycogenolysis in human muscle during epinephrine infusion has been investigated. The content of cAMP in resting muscle was 2.7 +/- 0.7 (SD) mumol . kg dry muscle-1 and increased threefold during the first 5 min of infusion. Total glycogen phosphorylase and glycogen synthase activities were unchanged during the infusion. The proportion of phosphorylase in the a form in the basal state was estimated to be at least 22.5% and during infusion 80-90%. During infusion, synthase I activity decreased. The muscle glycogen content was 340 mmol . kg dry wt-1 and decreased during the first 2 min of infusion at a rate of 11.0 mmol glycosyl units . kg dry wt-1 . min-1. Prolonged infusion resulted in a much lower glycogenolytic rate, even though most of the phosphorylase was still in the a form. Accumulation of hexose monophosphates and lactate followed the changes in glycogen. It was concluded that despite the almost total transformation of phosphorylase to the a form, the in vivo activity was maintained at a low level. It is suggested that this may be due to a low concentration of inorganic phosphate at the active site of the enzyme.

The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle.

The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle has been investigated using the needle biopsy technique. Preliminary studies showed that the activity of phosphorylase in vitro was dependent upon the concentration of inorganic phosphate (Pi) used in the assay system. The Km of phosphorylase a for Pi was found to be 26.2 mmol/l, and that of (a+b) (assayed in the presence of saturating AMP) was 6.8 mmol/l. Because of the difference in Km the apparent percentage of a to (a+b) activity varies with the Pi concentration used in the assay system. Phosphorylase a and (a+b) activities were therefore adjusted to saturating Pi concentrations. The ratio of the activities in this case is independent of the Pi concentration and constitutes a minimal estimate of the fraction of phosphorylase molecules in the a form. The fraction of phosphorylase in the a form in resting muscle was as a mean 22%. Despite nearly a quarter of the phosphorylase being in the a form glycogenolytic activity is extremely low. It is proposed that the concentration of Pi at the active site of the enzyme is low compared to the Km for this of either form of the enzyme, and is limiting to activity. A Pi concentration in resting muscle of 1-3 mmol/l was calculated. During epinephrine infusion at rest 90% of the phosphorylase was transformed to the a form but only a moderate increase in the glycogenolytic rate occurred. This rate approximated to 5-10% of the maximum rate of the enzyme (Vmaxa). During prolonged epinephrine infusion the glycogenolytic rate decreased despite the continuance of 90% or more of the phosphorylase in the a form. In contrast to epinephrine infusion prolonged ischemia resulted in a decrease in the mole fraction of phosphorylase a and simultaneously in an increase of the glycogenolytic rate. During isometric and dynamic exercise there was a rapid transformation of phosphorylase b to a paralleled by pronounced increase in the rate of glycogen breakdown. The increased rate of glycogenolysis during isometric exercise was close to the Vmax of phosphorylase a in vivo. When either form of exercise was continued to fatigue/exhaustion, a re-transformation of phosphorylase a to b was observed. During dynamic exercise cAMP in the muscle increased two fold. This increase was blocked by the prior administration of propranolol.+

Regulation of glycogenolysis in human muscle at rest and during exercise.

The regulation of glycogenolysis in human muscle during isometric and dynamic exercise has been investigated. Total glycogen phosphorylase and synthase activities were unchanged during exercise. The fraction of phosphorylase in the alpha form at rest was estimated to be 20%, but the data indicate that the in vivo activity was low and critically dependent on the concentration of inorganic phosphate (Pi) in the muscle. Phosphorylase alpha increased initially 2.4-fold during isometric contraction and 1.6-fold during maximal bicycle exercise but reverted to or below the resting value at fatigue/exhaustion. At rest synthase I was 17-48% of the total activity but decreased during exercise to about half of this value. The reciprocal changes in phosphorylase and synthase correlate with the enhanced rate of glycogenolysis during exercise. Michaelis constant (Km) for Pi was 27 mmol . l-1 for phosphorylase alpha and 7 mmol . l-1 for alpha + b. From consideration of the changes in Pi during exercise (to 20-30 mmol . l-1) it was concluded that Pi is one of the main factors determining phosphorylase activity and provides a link between phosphocreatine breakdown and glycogen utilization in muscle.