Purification of rat papillary collecting duct cells: functional and metabolic assessment.

Rat renal papillary collecting duct (PCD) cells were isolated using collagenase and hyaluronidase digestion and a three-step low-speed centrifugation. As assessed by binding of the lectin Dolichos biflorus and determination of vasopressin-sensitive adenylate cyclase and Na+-K+-ATPase, the enrichment of PCD cells over a crude papillary cell preparation was 1.8, 2.4, and 1.4, respectively. Microscopic evaluation indicated that the preparation was greater than 90% pure PCD cells. The isolated cells were viable as evident from the high K/Na ratio of intracellular electrolytes measured by electron probe analysis (5.3), from the high ATP/ADP ratio (2.15), and the metabolic response to alterations in Na transport. Exposure to 2 mM ouabain or removal of Na reduced O2 consumption by 25-35%; the uncoupler carboxylcyanide-m-chlorophenylhydrazone more than doubled O2 consumption. In the presence of 14 mM glucose and at a PO2 of 100 Torr the cells produced substantial quantities of lactate. This aerobic glycolysis may account for greater than 20% of the ATP production. In the presence of rotenone, glycolysis increased by 56% and was able to maintain the cellular ATP level at 65% of control. In the absence of any exogenous substrate PCD cells respired normally and had a close to normal ATP content, but lactate production was markedly decreased. These results demonstrate that viable PCD cells can be isolated from rat kidney. At normal PO2 and in the presence of D-glucose the cells show a substantial amount of aerobic glycolysis, although their mitochondrial respiration is not rate limiting. In the absence of glucose the cells derive the majority of their energy from an as yet unidentified endogenous substrate.

Assessment of techniques for preventing glycolysis in cardiac muscle.

Studies of myocardial function during ischaemic or anoxia would be assisted if it were possible to inhibit glycolysis completely and reversibly. Three methods of preventing glycolysis in isolated perfused ferret hearts were studied: (a) the removal of glucose from the perfusate followed by manoeuvres designed to deplete glycogen stores, (b) the use of 2-deoxyglucose, and (c) the use of iodoacetate. The rate of glycolysis before and after applying the three methods was assessed by measuring lactate production during short periods of anoxia. Metabolic changes associated with each method were investigated with phosphorus nuclear magnetic resonance, and other side effects associated with each method were assessed by measuring developed pressure. The results show that removal of glucose followed by glycogen depletion reduced the rate of glycolysis to any chosen extent and that the method was reversible. 2-Deoxyglucose treatment did not lead to complete inhibition of glycolysis, was not reversible, and reduced the concentrations of phosphocreatinine and adenosine triphosphate in the heart. Iodoacetate treatment completely blocked glycolysis, but it was not reversible and subsequent periods of anoxia lead to a rapid fall in adenosine triphosphate owing to the accumulation of phosphorylated glycolytic intermediates.

Physiologic assessment of phosphoglycerate mutase deficiency: incremental exercise test.

A third case of phosphoglycerate mutase (PGAM) deficiency, a metabolic myopathy involving terminal glycolysis, was identified in a 24-year-old black man with episodic, exercise-induced myoglobinuria since age 13. To better understand the physiologic consequences of PGAM deficiency, incremental exercise testing was performed. Results were compared with those of two patients having myophosphorylase deficiency and five normals. In contrast to the patients with phosphorylase deficiency, the PGAM-deficient patient achieved near-normal levels of maximal exercise and produced a normal peak lactate after exercise. The mechanisms underlying the asymptomatic performance of such strenuous exercise in this case are uncertain, but the data suggest that unidentified factors are operative in precipitating attacks of myoglobinuria in patients with some metabolic myopathies. Despite similar clinical histories, patients with different glycolytic enzyme deficiencies can have striking differences in exercise tolerance.

Energy cost of membrane depolarization in hog carotid artery.

Past studies have shown that during stable stepwise activations of vascular smooth muscle with varying concentrations of high-K+, both cytoplasmic free-Ca++ and membrane depolarization vary. On the other hand, during stepwise activations with varying concentrations of external Ca++ in the presence of constant external Ca++ in the presence of constant external depolarizing high-K+, cytoplasmic free-Ca++ varies, while membrane depolarization remains relatively constant. In this study, the rates at which suprabasal energy metabolism (oxygen consumption and lactic acid production) increased with increasing isometric tension maintenance were measured under both circumstances. Suprabasal energy metabolism with increasing membrane depolarization (increasing external K+) exceeded that with constant depolarizing-[K+] and varying Ca++ by less than 2.5% at all levels of activation, which was not statistically significant (P greater than 0.70). We conclude therefore that the steady state metabolic energy cost of membrane depolarization per se during contractile activity in vascular smooth muscle from a tonic conducting vessel (hog carotid artery) is negligible. Although the possibility cannot be excluded, we find no metabolic evidence that increased cytoplasmic free-Ca++ itself activates an ATPase associated with Ca++ sequestration and/or extrusion beyond that present in the relaxed state. Activation of hog carotid artery with an isosmotic K+-for-Na+-substituted medium fails to stimulate aerobic glycolysis at all levels of K+ substitution. Experiments were performed at the muscle length optimal for isometric tension generation and at 37 degrees C.

Platelet storage: an assessment of the requirements for plasma and oxygen.

Platelets from several units of plasma were pooled, and then resuspended either as platelet concentrate (PC) (60 ml) or as platelet rich plasma (PRP) (220 ml) and followed during 72 hours of storage at 22 C. Aggregation, pH, and hypotonic shock response are better maintained in the larger volume of plasma. However the decreased pH and function in the PC is not the result of lactate production. While the relative concentration, expressed in mg/dl, is higher in the PC, the absolute production per platelet and, therefore, the total amount of lactate is higher in PRP. Glucose levels are always sufficiently high to permit glucose to be used as a metabolic substrate even after 72 hours. PC maintained in nitrogen have better in vitro function than do platelets stored either in oxygen or CO2. CO2 can easily diffuse through the plastic bag; the same is not true of oxygen. Less than 15 per cent of the volume of oxygen introduced into the bag passed out through the PVC plastic over a 72-hour period. The data suggest that during storage, anaerobic glycolysis is the preferred metabolic route. The relatively poor performance of the PC maybe the result of limitation of some unidentified substrate or cofactor which is present in plasma and which is necessary for maintaining function.

The aerobic metabolism of porcine carotid artery and its relationship to isometric force. Energy cost of isometric contraction.

1. The O2 consumption and aerobic lactate production of porcine carotid artery have been studied in relation to active isometric tension maintenance in order to estimate the tension cost, i.e., ATP hydrolysis rate per unit force maintained. 2. The relationship between total metabolism and isometric force was measured at several muscle lengths in order to distinguish tension-dependent and -independent components of ATP utilization. 3. At the maximum isometric tension observed, 2 kg wt/wt/cm2, 1.3 mumole ATP/M in per gram wet weight out of a total metabolism of 2 mumole/min is tension de pardent. 4. The absolute levels of force, O2 consumption, lactate production and the ratio of oxidative to glycolytic metabolism varied considerably with the stimulant; however, the tension-dependent ATP utilization was invariant with respect to the mode of stimulation.

A physiological approach to the assessment of disease activity in rheumatoid arthritis.

A method is described for assessing the in vivo oxygen consumption and lactate production rates of human knee joints. It is based on the rate of fall of P(o2) and the rate of rise in lactate concentration in an intra-articular saline pool after interruption of the circulation to the joint with an arterial tourniquet. Studies in 5 control patients with degenerative joint disease and 29 patients with rheumatoid arthritis showed a 2- to 3-fold higher mean oxygen uptake rate and a 10- to 12-fold higher mean lactate appearance rate in the saline in the rheumatoid joints with severe disease compared to the control joints. These metabolic variables correlated with tissue metabolic demand as estimated in synovial biopsies. (133)Xe washout from the intra-articular space, which reflects joint circulatory flow, showed a 3-fold greater mean washout rate from the rheumatoid joints (48 studies) than control joints (7 studies) with extensive overlap between the two groups. (133)Xe washout rate correlated with knee joint inflammation estimated both clinically and histologically. After synovectomy in four patients, the operated knee showed a greater fall in lactate production than the opposite knee in three of these patients. Neither knee joint oxygen uptake nor (133)Xe washout rate changed significantly. Intra-articular corticosteroid injection (eight patients) resulted in decreased lactate production and a decreased (133)Xe washout rate in the injected knee and variable results in the untreated knee. Oxygen uptake again was unchanged after therapy.