Open-chest cardiac massage without major thoracotomy: metabolic indicators of coronary and cerebral perfusion.

OBJECTIVE: To compare the coronary and cerebral perfusion achieved using a novel method of minimally-invasive, direct cardiac massage to that obtained using bimanual, open-chest cardiac massage. DESIGN: Prospective, controlled animal study with repeated measures. SETTING: University research laboratory. SUBJECTS: Large domestic swine. INTERVENTIONS: Aortic, coronary sinus, jugular venous and pulmonary artery catheters were placed. Following an equilibration period, ventricular fibrillation was induced. After 4 min of untreated ventricular fibrillation, animals underwent bimanual, open-chest cardiac massage (N = 6) or minimally-invasive, direct cardiac massage using a novel device for direct cardiac compression (N = 6). Adrenaline was administered at a dose of 1 mg intravenously every 5 min. MEASUREMENTS: Systemic metabolic parameters, (arterial PO2, PCO2 and lactate concentration) and coronary sinus and jugular venous metabolic parameters (pH, PVO2, SVO2, PVCO2 and lactate concentration) were measured and calculated (coronary sinus/jugular-arterial SVO2, coronary sinus/jugular-arterial PCO2 and lactate differences) at baseline and at 10, 20 and 30 min following induction of ventricular fibrillation. Animals were euthanised after 30 min with no attempt at defibrillation. MAIN RESULTS: Oxygen tension and oxygen saturation of coronary sinus blood declined significantly during the experimental period, but no differences were noted between treatment groups. The coronary sinus-arterial oxygen saturation difference increased during the study with no significant differences between groups. Coronary sinus PCO2 and the coronary sinus-arterial PCO2 difference increased significantly in both experimental groups during cardiac massage. No inter-group differences were noted. A similar relationship was noted in coronary sinus lactate values. The coronary sinus-arterial lactate difference displayed a positive balance at all intervals with no differences noted between group values. The oxygen tension and oxygen saturation of jugular venous blood, were reduced from baseline levels with both treatments. The jugular-arterial oxygen saturation difference increased in both groups compared to baseline values. Between group values were significantly different only at the 20 min interval. Both the jugular venous PCO2 and the jugular-arterial PCO2 gradient were elevated at all intervals, but no inter-group differences were noted. Jugular venous lactate concentration rose steadily with time in both groups. No significant increase in the jugular-arterial lactate gradient was noted at any time point. CONCLUSIONS: Minimally-invasive, direct cardiac massage provides coronary and cerebral perfusion similar to that achieved using standard open-chest cardiac massage. This method may provide a more effective substitute for standard, closed-chest cardiac massage in cases of refractory cardiac arrest.

Extended intravascular shunting in an experimental model of vascular injury.

PURPOSE: To examine the extended patency (> 24 hrs) of heparin-bonded intravascular shunts in a porcine model of vascular injury. PROCEDURES: Adult swine underwent bilateral, common iliac artery resection (n = 5) or bilateral common iliac vein resection (n = 5) and vessel replacement with interposition, heparin-bonded shunts. Three control swine had vessel dissection only. Hematologic and coagulation profiles were measured at baseline and 24 hrs. Limb perfusion was assessed at 24 hrs by clinical exam and angiography. RESULTS: At 24 hrs, all limbs in both shunt groups were well perfused. All arterial shunts were angiographically patent. No distal emboli were detected. Nine of 10 venous shunts were patent, seven were lined with non-occluding thrombus. No alterations in hematologic or coagulation profiles were noted. CONCLUSIONS: Heparin-bonded shunts remained patent in arteries for 24 hours. Shunts placed in the venous system were prone to thrombus formation but most remained patent.

Phosphorylation of the carboxyl-terminal region of dystrophin.

Dystrophin is a protein product of the gene responsible for Duchenne and Becker muscular dystrophy. The protein is localized to the inner surface of sarcolemma and is associated with a group of membrane (glyco)proteins. Dystrophin links cytoskeletal actins via the dystrophin-associated protein complex to extracellular matrix protein, laminin. This structural organization implicates the role of dystrophin in stabilizing the sarcolemma of muscle fibers. Precisely how dystrophin functions is far from clear. The presence of an array of isoforms of the C-terminal region of dystrophin suggests that dystrophin may have functions other than structural. In agreement, many potential phosphorylation sites are found in the C-terminal region of dystrophin, and the C-terminal region of dystrophin is phosphorylated both in vitro and in vivo by many protein kinases, including MAP kinase, p34cdc2 kinase, CaM kinase, and casein kinase, and is dephosphorylated by calcineurin. The C-terminal domain of dystrophin is also a substrate for hierarchical phosphorylation by casein kinase-2 and GSK-3. These observations, in accordance with the finding that the cysteine-rich region binds to Ca2+, Zn2+, and calmodulin, suggest an active involvement of dystrophin in transducing signals across muscle sarcolemma. Phosphorylation-dephosphorylation of the C-terminal region of dystrophin may play a role in regulating dystrophin-protein interactions and (or) transducing signal from the extracellular matrix via the dystrophin molecule to the cytoskeleton.

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase.

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated, in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268:21901-21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44mpk Partial purification and immunological analysis show that a mammalian kinase related to p44mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.

Direct cardiac massage without major thoracotomy: feasibility and systemic blood flow.

BACKGROUND: Open-chest cardiac massage (OC-CM) provides higher blood pressure and flow than closed-chest compression and may improve the probability of successful resuscitation from cardiac arrest. Its clinical use has been limited by its requirement for a major thoracotomy. The present pilot study tested the technical feasibility of performing effective direct cardiac massage without a major thoracic incision, by using a simple, manually-powered plunger-like device, inserted through a small thoracic incision, to cyclically compress the cardiac ventricles. The method was termed minimally-invasive direct cardiac massage (MID-CM). Systemic blood flow using MID-CM was compared to that with OC-CM, by both direct systemic hemodynamic measurements, cumulative metabolic indicators of the ratio of whole body oxygen delivery and oxygen consumption, and a metabolic index of pulmonary blood flow. METHODS: In 12 large swine, baseline systemic and pulmonary hemodynamic measurements were performed. Arterial and mixed venous blood gases and metabolic indicators of systemic blood flow were measured. Ventricular fibrillation was induced and after 4 min, animals underwent either bimanual OC-CM (N = 6) or MID-CM (N = 6). At 10, 20 and 30 min, hemodynamic and metabolic measurements were repeated. RESULTS: Systemic Blood Pressure: Aortic systolic and diastolic blood pressures were reduced from baseline levels with both OC-CM and MID-CM. No difference in pressure was noted between OC-CM and MID-CM groups. Pulmonary Artery Pressure: Pulmonary artery systolic pressure was elevated from baseline during OC-CM and MID-CM. Pulmonary artery diastolic pressures remained constant throughout the resuscitation period in both groups. No differences in pulmonary systolic or diastolic pressure were noted between OC-CM and MID-CM groups. A trend towards higher pulmonary systolic pressures appeared with MID-CM. Thermodilution Blood Flow: Cardiac index fell from baseline levels with OC-CM and MID-CM. No difference in cardiac index was noted between OC-CM and MID-CM groups. Metabolic Indices: Mixed venous O2 saturation decreased from baseline levels during resuscitation in both experimental groups, with a further decrease at 30 min compared to 10- and 20-min levels. No difference was noted between OC-CM and MID-CM groups at any point. Arterial pH was reduced from baseline levels at 30 min in both groups compared to baseline but no difference was noted between groups.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of calcitonin gene-related peptide and nitric oxide in gastric mucosal hyperemia and protection.

It has been suggested that capsaicin-induced hyperemia and mucosal protection occurs via calcitonin-gene-related peptide (CGRP) release from gastric afferent sensory neurons and nitric oxide (NO)-mediated vasodilation. The purpose of this study was to determine whether capsaicin and/or bile acid induced hyperemia is mediated by CGRP and/or NO. Male Sprague-Dawley rats (280-350 g) were anesthetized, and the glandular stomach (blood supply intact) was chambered between two plastic rings. Animals were divided into six groups. Normal saline (groups 1 and 4), the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME; 3.75 mg/ml, groups 2 and 5), or the CGRP antagonist hCGRP8-37 (0.047 mg/ml, groups 3 and 6) was continuously infused intraarterially (ia) close to the stomach at a rate of 0.034 ml/min for 1 hr via a catheter inserted retrogradely into the splenic artery. Fifteen minutes after the onset of this infusion, the gastric mucosa was topically exposed to neutral saline solution for 15 min, followed by 160 microM capsaicin for 15 min. The mucosa was then injured by a 15-min exposure to either 5 mM acidified taurocholate (ATC, pH 1.2) in groups 1-3 or 10 mM ATC in groups 4-6. Gastric mucosal blood flow (ml/min/100 g) was continuously measured (laser doppler), and injury was assessed by measuring net transmucosal H+ flux, luminal accumulation of DNA, and histologic grading (0 = no injury to 3 = severe) by an independent observer. Intraarterial infusion of L-NAME significantly blocked the hyperemic response of topical capsaicin while having minimal effect on bile acid-induced hyperemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of dystrophin. The carboxyl-terminal region of dystrophin is a substrate for in vitro phosphorylation by p34cdc2 protein kinase.

In this paper, we report that p34cdc2 protein kinase phosphorylates recombinant fragments of skeletal muscle dystrophin with a maximal incorporation of 1.8 mol of Pi/mol of protein. Phosphorylation of both serine and threonine residues occurs within the carboxyl-terminal 201 amino acids of dystrophin, with phosphothreonine localized to within 25 residues of the carboxyl terminus. Supporting these in vitro studies, we also show that native dystrophin is phosphorylated by p34cdc2 kinase in isolated sarcolemmal vesicles. Sequence analysis indicates two consensus sites for p34cdc2 protein kinase within the carboxyl-terminal 201 amino acids of dystrophin. Importantly, neither of these sites is conserved in dystrophin-related protein, and only one site is conserved in the 71-kDa alternative product of the Duchenne muscular dystrophy gene, despite an otherwise extremely high degree of sequence conservation between these proteins. Importantly, in this study we also show that dystrophin is phosphorylated in vivo in rat skeletal muscle primary cultures, and we suggest that further investigation of both in vivo and in vitro phosphorylation of this protein will comprise an important part in determination of its function(s).

Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli.

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells.

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca(2+)-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca2+ 'storage' proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca(2+)-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca(2+)-binding proteins of muscle and nonmuscle cells, respectively. Other Ca(2+)-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca2+ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca2+ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.

Seasonal variations in the rate and capacity of cardiac SR calcium accumulation in a hibernating species.

The rate of calcium uptake and the level of calcium accumulation was measured in cardiac muscle SR from hibernating and nonhibernating Richardson's ground squirrels. In whole heart homogenates, the rate of calcium uptake was higher (P less than 0.05) in hibernating animals than it was in active animals. Further purification of homogenates into sacroplasmic reticulum (SR) preparations showed that the hibernating animals had the highest rate of calcium uptake and the greatest level of calcium accumulation. These results could not be explained by variations in non-SR membrane contaminants nor by changes in the maximal activity or total amount of a SR marker enzyme, the Ca(2+)-ATPase. The addition of ryanodine to the calcium uptake medium increased the level of calcium accumulation in all groups by a similar amount. It is concluded that the high rate of calcium uptake by isolated cardiac SR vesicles from hibernating ground squirrels reflects the activity of the organelle in vivo, and that the ability of the ryanodine-insensitive population of SR vesicles to accumulate calcium is affected by hibernation.

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Altered properties of calsequestrin and the ryanodine receptor in the cardiac sarcoplasmic reticulum of hibernating mammals.

A novel isoform of calsequestrin was identified in sarcoplasmic reticulum vesicles from myocardial tissue of two species of hibernating ground squirrel. The protein was identified as calsequestrin by its cross-reactivity with antibodies raised against bovine cardiac calsequestrin, its pH-sensitive mobility in sodium dodecylsulphate-polyacrylamide gels, staining blue with the cationic carbocyanine dye 'Stains-All', binding peroxidase-conjugated concanavalin A, its endoglycosidase F sensitivity. Its NH2-terminal amino acid sequence is similar, but not identical, to that already determined for cardiac calsequestrin. Some of the biochemical properties of this protein distinguish it from the other mammalian isoforms. It has a unique electrophoretic mobility in both alkaline and neutral sodium dodecylsulphate-polyacrylamide gel electrophoresis, it appears to have a molecular weight approximately 7% greater than that of cardiac calsequestrin from other mammalian species, and its glycosylation pattern differs. This novel form of calsequestrin is expressed in cardiac SR vesicles which possess an abnormally high number of Ca2(+)-release channel/ryanodine receptor molecules. This ryanodine receptor also shows an altered Ca2(+)-sensitivity of ryanodine binding. The divergent biophysical properties of this novel form of cardiac calsequestrin, together with the apparently atypical ryanodine receptors in the cardiac sarcoplasmic reticulum membranes may have some functional significance in the adaptive mechanisms which allow the heart to function despite the severely reduced body temperatures (to approx. 0 degree C) encountered during hibernation.

Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice.

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen-antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.

Cold-induced changes in uncoupling protein and GDP binding sites in brown fat of ob/ob mice.

The acute and chronic effects of cold exposure on the thermogenic activity and capacity of brown fat of obese (ob/ob) and lean mice have been examined. After 1 h at 13 degrees C, mitochondrial GDP binding increased in warm-acclimated (28 degrees C for 3 wk) lean mice, and increases in mitochondrial mass and the specific mitochondrial concentration of uncoupling protein were evident at 24 h. Long-term cold exposure led to further progressive increases in the specific mitochondrial concentration and total tissue content of uncoupling protein. The acute increase in GDP binding was due to an unmasking of binding sites on the protein and was accompanied by a parallel increase in the acetate-induced swelling of the mitochondria. In contrast to lean animals, ob/ob mice did not exhibit an increase in GDP binding or unmasking of binding sites during the first 24 h of cold exposure; unmasking was, however, evident by 3 days. A delay in the stimulation by cold of both the specific mitochondrial concentration of uncoupling protein (not increased until 3 days) and the mitochondrial content of brown fat (not increased until 8 days) occurred in the obese mice. Acute warm exposure (28 degrees C) of cold-acclimated (13 degrees C for 3 wk) mice led to a reduction in GDP binding in both lean and obese animals because of a rapid masking of binding sites. Mitochondrial content and the specific mitochondrial concentration of uncoupling protein fell progressively in both groups of mice with continued warm exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis.

In this article we comment on the various in vitro biochemical measurements employed to assess the thermogenic activity and capacity of brown adipose tissue. The meaning and significance of changes in tissue weight, protein content, cell number, and mitochondrial mass are each summarized. In addition, various indices of the proton conductance pathway-mitochondrial swelling, proton conductance, uncoupling protein concentration, and GDP binding studies--are discussed. The issue of unmasking and masking of GDP binding sites is reviewed; recent reports have clearly demonstrated unmasking and masking, and it is concluded that GDP binding studies are an index of the activity of uncoupling protein, rather than a measure of its concentration. It is suggested that tissue mass, mitochondrial content, mitochondrial GDP binding, and uncoupling protein concentration represent core measurements for the biochemical assessment of the thermogenic activity and capacity of brown adipose tissue. Auxiliary measurements include Scatchard analysis of GDP binding data to distinguish changes in the number of binding sites from potential changes in Kd, and mitochondrial swelling studies, as an additional index of proton permeability. The distinction between thermogenic activity (GDP binding, proton permeability) and capacity (uncoupling protein content), both on a per unit of mitochondrial protein and per tissue basis, is emphasized.

Brown fat thermogenesis during hibernation and arousal in Richardson's ground squirrel.

The thermogenic activity [mitochondrial guanosine 5'-diphosphate (GDP) binding] and capacity (uncoupling protein concentration, cytochrome oxidase activity) of brown adipose tissue have been investigated at different phases of the seasonally linked hibernation cycle in Richardson's ground squirrel. The amount of axillary brown adipose tissue and the total mitochondrial content of the tissue were substantially greater in hibernating squirrels than in squirrels caught posthibernation in April or May; cold acclimation induced qualitatively similar differences. The specific mitochondrial concentration of uncoupling protein was high under all conditions (compared with other species), differing little between hibernating, posthibernating, and cold-acclimated squirrels. The thermogenic capacity of brown adipose tissue in Richardson's ground squirrels is therefore modulated almost exclusively by changes in the mitochondrial content of the tissue. Mitochondrial GDP binding was increased on cold acclimation, but similar binding levels were observed in hibernating and posthibernation (May) animals. GDP binding and the GDP-sensitive component of acetate-induced mitochondrial swelling were increased during the early stages of arousal from hibernation. These changes, which indicate an activation of the thermogenic proton conductance pathway in arousal, occurred without an alteration in the specific mitochondrial concentration of uncoupling protein. Increased GDP binding during arousal is clearly due to the unmasking of binding sites, reflecting an acute activation of preexisting uncoupling protein.

Evidence that the acute unmasking of GDP-binding sites in brown adipose tissue mitochondria is not dependent on mitochondrial swelling.

The role of mitochondrial swelling in the unmasking of GDP-binding sites on brown adipose tissue mitochondria has been examined in mice. Acute cold exposure (6 degrees C for 1 h) led to increases in GDP binding without changes in the concentration of uncoupling protein, indicating that an unmasking of binding sites had occurred. Measurements of mitochondrial matrix volume suggested that an acute unmasking of GDP-binding sites took place without swelling of the mitochondria. In addition, the induction of a rapid preswelling of the mitochondria by incubation in KCl in the presence of valinomycin did not affect the cold-induced unmasking of GDP-binding sites. It is concluded that the acute unmasking of GDP-binding sites on uncoupling protein in brown adipose tissue is not due simply to mitochondrial swelling.

Acute effects of a beta-adrenoceptor agonist (BRL 26830A) on rat brown-adipose-tissue mitochondria. Increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein.

GDP binding, proton conductance and the specific concentration of uncoupling protein were measured in brown-adipose-tissue mitochondria of rats treated acutely with the novel beta-agonist, BRL 26830A. At 1 h after dosing with BRL 26830A, mitochondrial GDP binding was increased more than 2-fold. The increase in binding resulted from an increase in the number of binding sites. An iterative analysis of Scatchard binding data suggested that there is only one high-affinity GDP-binding site (Kd 0.3 microM) in brown-adipose-tissue mitochondria. The acute increase in GDP binding produced by treatment with BRL 26830A occurred without any alteration in the specific mitochondrial concentration of uncoupling protein, as determined by radioimmunoassay. Treatment with the beta-agonist did, however, lead to a small increase in the GDP-sensitive component of mitochondrial proton conductance. These results indicate that GDP-binding sites on uncoupling protein can be rapidly unmasked after treatment with a brown-fat-specific beta-agonist, and that the increase in binding reflects an increase in the activity of the mitochondrial proton-conductance pathway.