Regional anaesthesia and pain management.

Despite recent advances in analgesia delivery techniques and the availability of new analgesic agents with favourable pharmacokinetic profiles, current evidence suggests that postoperative pain continues to be inadequately managed, with the proportion of patients reporting severe or extreme postoperative pain having changed little over the past decade. Regional techniques are superior to systemic opioid agents with regards to analgesia profile and adverse effects in the context of general, thoracic, gynaecological, orthopaedic and laparoscopic surgery. Outcome studies demonstrate that regional analgesic techniques also reduce multisystem co-morbidity and mortality following major surgery in high risk patients. This review will discuss the efficacy of regional anaesthetic techniques for acute postoperative analgesia, the impact of regional block techniques on physiological outcomes, and the implications of acute peri-operative regional anaesthesia on chronic (persistent) postoperative pain.

The effects of different analgesic regimens on transcutaneous CO2 after major surgery.

Ventilatory impairment may be detected by a rise in transcutaneous carbon dioxide levels (PtcCO(2)). This observational study assessed the clinical utility of PtcCO(2) monitoring in the postoperative period, and quantified the effect of different peri-operative analgesic regimens on postoperative respiratory function. Following pre-operative baseline PtcCO(2) recording, continuous PtcCO(2) monitoring was performed in 30 patients after major colorectal surgery for up to 24 h. Mean postoperative values of PtcCO(2) were 1.3 kPa (95% CI 1.0-1.5) higher than pre-operative values (p < 0.001). Patients receiving intravenous opioid patient controlled analgesia had a significantly higher elevation in postoperative PtcCO(2) compared to patients receiving epidural infusion analgesia, 1.8 kPa (CI 1.5-2.1) vs 0.7 kPa (CI 0.5-0.9) respectively (p < 0.001). The mean rise in PtcCO(2) following a single intravenous bolus of morphine delivered via PCA was 0.05 kPa (SEm 0.01), peaking at 12 min post-dose. The transcutaneous capnometer successfully recorded data for 98% of the total time it was applied to patients.

The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues.

The pyruvate, NAD(+)-isocitrate and 2-oxoglutarate dehydrogenases are key regulatory enzymes in intramitochondrial oxidative metabolism in mammalian tissues, and can all be activated by increases in Ca2+ in the micromolar range. There is now mounting evidence that hormones and other stimuli which act by increasing cytosolic Ca2+ also, as a result, cause increases in mitochondrial matrix Ca2+ and hence activation of these enzymes, suggesting that the primary physiological function of mitochondrial Ca2(+)-transport is to be involved in this relay mechanism. This may also explain how in such circumstances rates of ATP production may be increased to meet the greater demand, but without any decreases in ATP/ADP occurring.

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat-kidney mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.

Studies on mitochondrial Ca2+-transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria.

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.

Parallel increases in rates of fatty acid synthesis and in pyruvate dehydrogenase activity in isolated rat hepatocytes incubated with insulin.

The effect of insulin on the activity of pyruvate dehydrogenase is studied in isolated hepatocytes from fed rats. Insulin increases the 'initial' activity of pyruvate dehydrogenase by 30% without modifying the total activity of the enzyme. The maximal increase is reached 3 min after addition of the hormone and is dose-dependent. Insulin also increases the rate of fatty acid synthesis.

Control of glycogen synthesis by glucose, glycogen, and insulin in cultured human muscle cells.

A key feature of type 2 diabetes is impairment in the stimulation of glycogen synthesis in skeletal muscle by insulin. Glycogen synthesis and the activity of the enzyme glycogen synthase (GS) have been studied in human myoblasts in culture under a variety of experimental conditions. Incubation in the absence of glucose for up to 6 h caused an approximately 50% decrease in glycogen content, which was associated with a small decrease in the fractional activity of GS. Subsequent reincubation with physiological concentrations of glucose led to a dramatic increase in the rate of glycogen synthesis and in the fractional activity of GS, an effect which was both time- and glucose concentration-dependent and essentially additive with the effects of insulin. This effect was seen only after glycogen depletion. Inhibitors of signaling pathways involved in the stimulation of glycogen synthesis by insulin were without significant effect on the stimulatory action of glucose. These results indicate that at least two distinct mechanisms exist to stimulate glycogen synthesis in human muscle: one acting in response to insulin and the other acting in response to glucose after glycogen depletion, such as that which results from exercise or starvation.

Effects of tumor necrosis factor-alpha on insulin action in cultured human muscle cells.

Reported discrepancies in the effects of tumor necrosis factor (TNF)-alpha in modulating insulin sensitivity of cultured cells may relate both to cell types studied and to the time course of exposure to the cytokine. Additionally, the relationship of effects on glucose metabolism to changes in the insulin signaling pathway cannot be assumed. For in vitro study, the cell type most relevant to insulin resistance in humans is the cultured human muscle cell. In the present study, TNF brought about no change in the rate of glycogen synthesis in cultured human muscle cells unless present during differentiation. The presence of TNF (5 ng/ml) during the process of differentiation of myoblasts into mature myotubes diminished the response of glycogen synthesis to acute insulin stimulation. This finding was associated with an impairment of differentiation-dependent increases in total cellular glycogen synthase (GS) activity. Under the same conditions of TNF exposure, there was no effect on the response to acute insulin stimulation of the fractional activity of GS. Similarly, there was no effect on the insulin stimulation of protein kinase B (PKB) and inhibition of glycogen synthase kinase 3 (GSK-3). Acute insulin stimulation brought about a 4.08 +/- 0.44-fold stimulation of activity of PKB in the absence of TNF, with 4.81 +/- 0.70-fold stimulation in cells exposed to TNF. GSK-3 activity decreased to 74.0 +/- 5.8% of basal after insulin stimulation without TNF and 78.3 +/- 5.0% after TNF exposure. However, differentiation of myocytes, as defined by an increase in the acetylcholine receptor, myogenin, and mature creatine kinase isoform expression, was impaired in TNF-treated cells. These studies demonstrate that TNF, if present during differentiation, decreases insulin-stimulated rates of storage of glucose as glycogen and total GS activity but does not downregulate the insulin-signaling system to GS. More generally, TNF also inhibits differentiation of human muscle cells in culture.

Regulation of energy substrate metabolism in the diabetic heart.

The effects of diabetes on myocardial metabolism are complex in that they are tied to the systemic metabolic abnormalities of the disease (hyperglycemia and elevated levels of free fatty acid and ketone bodies), and changes in cardiomyocyte phenotype (e.g., down-regulation of glucose transporters and PDH activity). The cardiac adaptations appear to be driven by the severity of the systemic abnormalities of the disease. The diabetes-induced changes in the plasma milieu and cardiac phenotype both cause impaired glycolysis, pyruvate oxidation, and lactate uptake, and a greater dependency on fatty acids as a source of acetyl CoA. Studies in isolated hearts suggest that therapies aimed at decreasing fatty acid oxidation, or directly stimulating pyruvate oxidation would be of benefit to the diabetic heart during and following myocardial ischemia.

Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions.

It is now clear that the availability of different metabolic substrates can have a profound influence on the extent of damage incurred during episodes of cardiac ischaemia, and on cardiac functional recovery on reperfusion following ischaemia. In particular, increases in fatty acid availability and oxidation, compared to glucose oxidation, under such conditions leads to a worsening of outcome. Therefore metabolic interventions aimed at enhancing glucose utilisation and pyruvate oxidation at the expense of fatty acid oxidation is a valid therapeutic approach to the treatment of myocardial ischaemia. In particular, the development of agents which will promote full glucose oxidation as opposed to glycolysis alone, offer clear advantages. This can be accomplished by different means, including direct or indirect inhibition of CPT-I or inhibition of fatty acid beta-oxidation, or by direct or indirect activation of PDH. It is not yet clear which of these approaches offers the best treatment of cardiac ischaemia. To date, trimetazidine and carnitine have received limited approval in Europe for the treatment of angina; large scale clinical trials with the other agents mentioned above have not been completed. The increasing availability of agents affecting these specific sites, and the increasingly sophisticated techniques for assessing myocardial metabolism, should allow elucidation of the optimum metabolic targets and development of novel pharmacological agents for the treatment of ischaemic heart disease.

Cardioprotective effects of ranolazine (RS-43285) in the isolated perfused rabbit heart.

OBJECTIVE: The aim was to examine the putative cardioprotective effects of the novel antianginal agent, ranolazine, using an isolated rabbit heart model of ischaemia and reperfusion. METHODS: Hearts from male New Zealand White rabbits were perfused in the Langendorff mode with a recirculating Krebs buffer at a constant flow of 20-25 ml.min-1. After equilibration, hearts were treated with ranolazine (10 or 20 microM) or vehicle control for 10 min before exposure to a 30 min period of global ischaemia and 60 min reperfusion; a normoxic control group was also studied. Haemodynamic variables (left ventricular pressure), myocardial creatine kinase, and potassium release were measured at baseline (preischaemic) and at selected points during reperfusion; tissue calcium and ATP content were also measured and electron microscopy was performed. RESULTS: Left ventricular developed pressure during reperfusion was improved (p < 0.05) in a concentration dependent manner by 10 and 20 microM ranolazine (the baseline value was unaffected) with the latter dose resulting in a return to preischaemic values. The release of creatine kinase and potassium was reduced in the ranolazine groups (p < 0.05). A 2.5-fold increase in tissue calcium content in vehicle treated hearts at the end of reperfusion (compared to normoxic time control) was reduced by 10 microM ranolazine (p < 0.05) and completely prevented by 20 microM ranolazine. Similarly, the decrease in tissue ATP was largely inhibited by ranolazine in a concentration dependent manner. Electron microscopy showed that 20 microM ranolazine prevented the occurrence of many indications of reperfusion injury observed in vehicle treated control hearts, for example, blurring of myofibrillar Z bands, derangement of myofibrillar architecture, disruption of mitochondrial cristae and matrices, and the appearance of electron-dense bodies within them. The deposition of lanthanum chloride, a marker of blood vessel integrity, is also modified in the ranolazine treated hearts. CONCLUSIONS: Ranolazine has impressive cardioprotective properties in an isolated rabbit heart model of ischaemia and reperfusion, suggesting that the drug warrants further research into its precise mechanism of action.

The calcium sensitive dehydrogenases of vertebrate mitochondria.

Three important dehydrogenases in vertebrate mitochondria are activated by Ca2+ ions with half-maximal effects at about 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated within intact mitochondria when extramitochondrial Ca2+ is increased within the range of concentrations generally considered to occur in the cytoplasm of vertebrate cells. It is argued that the main role of the calcium transport system in the inner membrane of vertebrate mitochondria is to relay changes in the cytoplasmic concentration of Ca2+ into the mitochondrial matrix. In this way, hormones and other extracellular stimuli which stimulate ATP-requiring processes such as contraction and secretion through increases in the cytoplasmic concentration of Ca2+ may also increase intramitochondrial oxidative metabolism and hence the replenishment of ATP.

Evidence that adrenaline activates key oxidative enzymes in rat liver by increasing intramitochondrial [Ca2+].

The effects of intramitochondrial Ca2+ on the activities of the Ca2+-sensitive intramitochondrial enzymes, (i) pyruvate dehydrogenase (PDH) phosphate phosphatase, and (ii) oxoglutarate dehydrogenase (OGDH), were investigated in intact rat liver mitochondria by measuring (i) the amount of active PDH (PDHa) and (ii) the rate of decarboxylation of alpha-[l-14C]oxoglutarate (at non-saturating [oxoglutarate]), at different concentrations of extramitochondrial Ca2+. In the presence of Na+ and Mg2+, both PDH and OGDH could be activated by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM). When liver mitochondria were prepared from rats treated with adrenaline, and then incubated in Na-free media containing EGTA, both PDH and OGDH activities were found to be enhanced. Evidence is presented that the activation of these enzymes by adrenaline is brought about by a mechanism involving increases in intramitochondrial [Ca2+].

Effect of phenylephrine on pyruvate dehydrogenase activity in rat hepatocytes and its interaction with insulin and glucagon.

In isolated rat hepatocytes phenylephrine promotes a rapid increase in the amount of pyruvate dehydrogenase present in its active form (PDHa). This action is mediated by alpha 1-adrenergic receptors and is not observed in Ca2+-depleted hepatocytes. It is mimicked by the Ca2+ ionophore A23187. No changes in metabolites known to affect PDH activity are measured 3 min after addition of phenylephrine. Glucagon also increases PDHa, its action is additive to that of phenylephrine. The action of phenylephrine on PDHa could be explained by an increase in mitochondrial free Ca2+.

Hormonal regulation of fluxes through pyruvate dehydrogenase and the citric acid cycle in mammalian tissues.

Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. Activation can also be demonstrated within permeabilized and intact mitochondria. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+. In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP. In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism.

Pharmacological approaches to inhibit endogenous glucose production as a means of anti-diabetic therapy.

The inappropriate overproduction of glucose by the liver is one of the key contributors to the hyperglycaemia of the diabetic state, and thus is a logical site of intervention for novel anti-diabetic approaches. Metformin is the only currently marketed anti-hyperglycaemic drug whose action is attributed largely to its having inhibitory effects on hepatic glucose production, but its molecular site and mechanism(s) of action remain unknown, whereas the liver acting PPAR alpha agonists have their effects primarily on lipid metabolism. This review therefore rather focuses on candidate molecular targets within the liver for anti-hyperglycaemic therapy, and describes potential rate-controlling receptors and enzymes within the glucose producing pathways (glycogenolysis and gluconeogenesis). Most focus is directed towards inhibitors of the enzymes glucose-6-phosphatase, fructose-1,6-bisphosphatase and glycogen phosphorylase, and towards glucagon receptor antagonists, as these appear to be the most advanced in preclinical and clinical development, although progress with other potential targets is also described. Evidence of the anti-diabetic potential of such agents from animal studies is presented, and the relative merits of each approach are reviewed and compared. It is likely that such agents will become important additions to the therapeutic approaches to combat diabetes.

Uptake of 3H-deoxyglucose as a microassay of human neutrophil and monocyte activation.

The accumulation of 2-deoxy-D-[1-3H]glucose, a non-metabolised analogue of glucose provides a quantitative measurement of the state of activation of phagocytic cells. A microassay for 3H-deoxyglucose uptake by human neutrophils and monocytes is described. Optimal conditions for the assay include the use of 5 X 10(5) cells and 0.78 microCi/ml of deoxyglucose in a final volume of 0.2 ml per microtitre well, and an incubation time of 30 min at 37 degrees C. This simple, rapid and reproducible technique may find application in experimental immunology.

Protective effects of ranolazine in guinea-pig hearts during low-flow ischaemia and their association with increases in active pyruvate dehydrogenase.

1. In isolated Langendorff-perfused, electrically-paced, hearts of guinea-pigs, global low-flow-ischaemia (LFI; at 0.7 ml min-1) resulted in marked increases in the rates of release of lactate, lactate dehydrogenase (LDH) and creatine kinase (CK) over a 30 min period. At the end of the LFI period, tissue ATP content was significantly reduced from a control value of 11.8 +/- 0.8 (5) to 5.6 +/- 0.8 (5) mumol g-1 dry weight. 2. The presence of ranolazine [(+/-)-N-(2,6-dimethyl-phenyl)-4[2-hydroxy-3-(2-methoxy-phenoxyl)- propyl] - l-piperazine acetamide dihydro-chloride; RS-43285-193] at 10 microM, from 20 min prior to and during LFI, resulted in significant reductions in the release of lactate, LDH and CK during the ischaemic period and a significant preservation of tissue ATP (9.0 +/- 1.1 (6) mumol g-1 dry wt.). Ranolazine did not prevent the reductions in creatine phosphate or glycogen observed in LFI, nor did it have any significant effects on any contractile parameters before or during the LFI period. 3. Neither ranolazine nor LFI affected the total amounts of tissue pyruvate dehydrogenase (PDH) activity; however, the significant reduction in the amount of active, non-phosphorylated PDH caused by LFI (from 88.2 +/- 5.5 to 44.2 +/- 3.2% of total activity) was partially but significantly prevented by ranolazine (67.2 +/- 6.8%). This effect of ranolazine on PDH may be part of the mechanism whereby the compound reduces lactate release and preserves tissue ATP during ischaemia.