Creatine Analogue Research Articles

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibres. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22°C, containing 11 mM glucose as substrate, and supplemented with either 20 mM β-guanidinopropionic acid (β-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31P-NMR. Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, β-GPA-P, GA-P and GA-P over a similar time course (∼ 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK. Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K m and increased V max vs control), but none by GA, mGPA or β-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes.

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.

Cyclocreatine transport and cytotoxicity in rat glioma and human ovarian carcinoma cells: 31P-NMR spectroscopy.

Cyclocreatine (CY), an analogue of creatine, inhibits tumor growth in vivo and proliferation of tumor cells in vitro. The goal of this study was to probe the mechanism of CY transport and cytotoxicity in C6 rat glioma cells and OC238 human ovarian carcinoma cells (creatine kinase activities of 0.16 and 0.016 units/mg protein, respectively). In both cell lines, CY significantly inhibited cell growth with no effect on membrane integrity and on the content of nucleoside triphosphates. An intrinsic 31P-nuclear magnetic resonance (31P-NMR) signal of phosphocreatine, as well as accumulation of phosphocyclocreatine (PCY) after addition of CY, was observed for C6 glioma but not for the OC238 cells. Transport of CY in C6 glioma showed Michaelis-Menten kinetics for an active sodium-dependent component. Transport was reduced more than fivefold in low-glucose medium. The toxicity of CY to C6 glioma cells may be due to PCY accumulation and cellular swelling. Another mechanism must be invoked to explain CY effects on the human ovarian cancer cells in which no PCY accumulation could be detected and no cellular swelling was observed.

Creatine analogue beta-guanidinopropionic acid alters skeletal muscle AMP deaminase activity.

Dietary supplementation of the creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases in vitro skeletal muscle AMP deaminase (AMP-D) activity in rats. Downregulation of AMP-D activity was progressive and greater in fast-twitch muscles (70-80%) than in the slow-twitch soleus muscle (approximately 50%). The loss in AMP-D activity had little effect on inosine 5'-monophosphate accumulation in mixed-fiber muscle with intense tetanic contractions. In contrast, inosine 5'-monophosphate formation was evident earlier in fast-twitch red and white fiber sections of creatine-depleted animals during intense twitch contractions, indicating that fast-twitch muscle of beta-GPA-treated rats buffers decreases in the ATP/ADPfree ratio via deamination, even though AMP-D activity is less. Isoforms of skeletal muscle AMP-D mRNAs in mixed-fiber muscle were not altered by feeding beta-GPA for up to 9 wk. Creatine depletion did not alter total immunoreactivity; however, a redistribution of AMP-D immunoreactivity from primarily an approximately 80-kDa form toward lower apparent molecular mass species (approximately 60 and approximately 56 kDa) was observed. Posttranslational changes in AMP-D appear related to changes in activity.

NMR microscopy studies of tumor microenvironments

The microenvironmental heterogeneity, characteristic of solid tumors 500 üm in diameter or larger, arises from insufficient nutrient supply due to tumor growth exceeding the rate of vascularization. Multicellular spheroids were proposed as an in vitro experimental model system for simulating these stress environments. As in hypoxic regions of tumors, inner cell layers in spheroids show resistance to radiation and conventional chemotherapy, as well as enhanced expression of the angiogenic vascular endothelial growth factor (VEGF). We have applied NMR microscopy for the study of the effects of these microenvironments on tumor physiology.NMR microscopy experiments were developed for mapping gradients in water diffusion and metabolite content across intact, perfused spheroids (1 mm in diameter). These methods were tested in a study of the cytotoxic effects of cyclocreatine, a synthetic analogue of creatine. In contrast with most of the common anti cancer drugs, accumulation of cyclocreatine was observed across the entire viable rim of C6 glioma spheroids.

Preincubation with creatine enhances levels of creatine phosphate and prevents anoxic damage in rat hippocampal slices.

We have investigated the relationship between energy metabolism, NMDA-receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. The concentrations of energy metabolites were measured by ion-exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose-dependently (0.03-3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia-induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with (R,S)-3-guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia-induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK-801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.

Microtubule stabilization and potentiation of taxol activity by the creatine analog cyclocreatine.

Creatine kinase (CK), a key enzyme of cellular energetics, has been implicated in tumorigenesis. Cyclocreatine (CCr), which forms a stable phosphagen with a reduced rate of ATP regeneration through CK, inhibits the growth of many solid tumors. We report that CCr induces the formation of unusually stable microtubules that resist depolymerization by nocodazole. By reducing ATP availability, CCr may modulate the activity of kinases that regulate microtubule dynamics. Further, combinations of CCr and taxol resulted in the synergistic killing of breast tumor cells indicating that CCr may be a useful addition to chemotherapy's that include taxanes.

Increased growth of brown adipose tissue but its reduced thermogenic activity in creatine-depleted rats fed β-guanidinopropionic acid

To study the responses of thermogenic activity in brown adipose tissue (BAT) to creatine depletion, male Wistar rats were fed creatine analogue β-guanidinopropionic acid (β-GPA) for about 10 weeks. Compared to control rats, a marked decrease in the levels of high-energy phosphates, such as phosphocreatine and ATP, was noted in BAT of β-GPA rats. Conversely, upward trends in other chemical components (DNA, glycogen, and total protein) in BAT as well as an increase in BAT mass were observed in β-GPA rats, suggesting a tendency to hyperplasia of the BAT. The thermogenic activity (which was assessed by guanosine 5′-diphosphate binding to BAT mitochondria) in the mitochondria recovered from BAT of β-GPA rats, however, was not increased in response to such changes but rather decreased. Moreover, uncoupling protein (UCP) content in the mitochondrial fraction of β-GPA rats was significantly lower than that in control rats (the relative amounts were 77 ± 6 and 100 ± 4%, respectively). Nevertheless, surprisingly, the level of UCP mRNA was remarkably greater in β-GPA rats than in control rats. These observations indicate that there is a discordance between BAT growth and activity in β-GPA rats, thereby suggesting that a failure on and after UCP translation may be involved in the impairment of BAT thermogenic activity with creatine depletion. The impairment of BAT thermogenic activity, that is, UCP activity may indicate that uncoupling or heat production was inhibited in order to increase the ATP synthesis in BAT of β-GPA rats in compensation for a reduction in the levels of high-energy phosphates (including ATP), with resultant hypothermia.

Creatine Kinase Equilibration Follows Solution Thermodynamics in Skeletal Muscle.: 31P NMR STUDIES USING CREATINE ANALOGS

The hypothesis tested was whether creatine kinase (CK) equilibrates with its substrates and products in the cytosol as if in solution. We used the creatine analogs cyclocreatine (cCr) or beta-guanidopropionate (beta GPA) to test if mass action ratios (gamma) for CK in muscle could be predicted from combined equilibrium constants (Kcomb) measured in solutions mimicking the intracellular environment. Mice were fed cCr or beta GPA and their muscles assayed for substrates and products of the CK reaction by 31P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, gamma was indistinguishable from Kcomb in cCr-treated muscles demonstrating both PCr/Cr and phospho-analog/analog must have equilibrated with a constant and uniform cellular ATP/ADP ratio. In beta GPA-treated muscles, gamma was smaller than Kcomb due to a higher content of muscle beta GPA. Feeding beta GPA for 9-12 weeks resulted in a closer agreement between Kcomb and gamma, suggesting ATP/ADP ratios are not uniform within the muscle perhaps due to transient metabolic stress in some cells. From this analysis it follows that calculation of free ADP from the CK equilibrium for a heterogeneous population of cells with respect to total Cr and ATP content is correct only if chemical potentials of these cells are uniform.

Effects of creatine and β-guanidinopropionic acid on the growth of Ehrlich ascites tumor cells: i.p. injection and culture study

Growth of Ehrlich ascites tumor (EAT) cells in the abdominal space of mice or in cell culture was studied in response to i.p. injection or addition, respectively, of creatine or creatine analogue β-guanidinopropionic acid (β-GPA). The increase in body weight of the mice due to cancer growth was less in the β-GPA-injected than in the creatine- or sham-injected group. The volume of abdominal ascites and total cell counts at 11th day after implantation of EAT cells was significantly less in the β-GPA than in the other groups. The proliferation rate of EAT cells in the β-GPA group was 27% and 35% of the creatine- and sham-injected groups, respectively. Supplementation of creatine tended to enhance the growth of EAT cells. The creatine concentration in ascites fluid was approximately 4-times greater than in blood plasma of sham-injected control mice. But the creatine content in EAT cells was significantly reduced to approximately 50% in response to β-GPA injection. Cell culture without creatine caused a significant decrease in viability. The viability was improved, however, by addition of either creatine or serum into the medium. By contrast, it was not significantly increased by addition of serum alone which caused only a minor elevation of the creatine level (23 μM). It is suggested that EAT cell growth is inhibited by lowering the availability of creatine in association with some unknown factors in serum or ascites fluid.

Energetic status and mitochondrial oxidative capacity of rat skeletal muscle in response to creatine analogue ingestion

A creatine analogue, β-guanidinopropionic acid (α-GPA), was administered in the food (1% w/w) of 8 male rats for 6 weeks, while 8 control rats received a standard diet. Mitochondrial oxidative capacity and cytosolic modulators of mitochondrial oxidative phosphorylation (free ADP, ATP-to-free ADP ratio) were evaluated in the soleus and extensor digitorum longus (EDL) muscles. Mitochondrial adaptation to the diet was significantly different between muscles. Citrate synthase activity and mitochondrial ATP synthesis rate were 35 and 45% higher in EDL muscle, respectively, whereas they were virtually unchanged in the soleus muscle. In both muscles, 3-hydroxyacyl-CoA dehydrogenase activity remained unaffected. Regardless of muscle type, creatine, phosphocreatine and ATP concentrations, as well as the total adenine nucleotide content (ATP + ADP + AMP), were significantly lower in β-GPA fed rats. Whereas free ADP concentration remained unchanged, a significantly greater decrease in ATP-to-free ADP ratio was observed in EDL than in the soleus muscle. It is suggested that regulation of mitochondrial oxidative phosphorylation, through changes in metabolite concentrations, could be an important factor to consider for mitochondrial adaptation induced by β-GPA feeding.

Metabolite utilization and compartmentation in porcine carotid artery: a study using beta-guanidinopropionic acid

The relationship between substrate and metabolism in vascular smooth muscle has been investigated by studying the acute energetic effects caused by the creatine analogue beta-guanidinopropionic acid (beta-GPA) on porcine carotid arteries using 31P-nuclear magnetic resonance (NMR). Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 50 mM beta-GPA, and either 11 mM glucose or 5 mM pyruvate as substrate. beta-GPA enters the cells and becomes phosphorylated by creatine kinase to produce beta-GPA-P. Perfusion with beta-GPA leads to the formation of NMR observable beta-GPA-P (after 2.5 h). The appearance of beta-GPA-P with time was significantly greater when glucose was used as substrate. To differentiate between oxidative and glycolytic metabolism in the phosphorylation of beta-GPA, 1 mM cyanide was included in the perfusion buffer containing 50 mM beta-GPA and 11 mM glucose. No phosphocreatine (PCr) was observed with these conditions, and there was a small but significant decrease in ATP concentration ([ATP]) compared with glucose perfusion without cyanide (0.56 +/- 0.02 to 0.47 +/- 0.02 mumol/g wet wt), that was greater than the concentration with pyruvate as substrate (0.25 +/- 0.03 mumol/g wet wt). Thus the [ATP] during cyanide treatment is maintained with glycolytic metabolism. Despite the relatively high [ATP], accumulation of beta-GPA-P only occurred over a much slower time course ( > 10 h) than without cyanide.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of contractile properties of extensor digitorum longus in response to creatine-analogue administration and/or hindlimb suspension in rats.

Changes of contractile properties of extensor digitorum longus in response to hindlimb suspension and/or altered high-energy phosphate contents were studied in rats. A reduction of high-energy phosphates, especially phosphocreatine, was seen in rats fed creatine analogue beta-guanidinopropionic acid (beta-GPA), but they were elevated after 10-d supplementation of creatine. The one-half relaxation time was increased by feeding beta-GPA, but was normalized by creatine supply. The fatigue resistance of creatine-depleted muscle was significantly improved, but tended to decrease by suspension and creatine supply, although it was still better than that in the control diet group. It is indicated that the contractile properties of muscle are influenced by the high-energy phosphate content. It is also suggested that the endurance capacity may be influenced by the mitochondrial respiratory capacity, but not necessarily by the levels of high-energy phosphates.

Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle.

This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid [creatine depletion juvenile (CDJ) group]. Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens [normal diet adult and creatine depletion adult (CDA) groups]. After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC distribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC transitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of animals to be used.

The Cloning and Expression of a Human Creatine Transporter

A human creatine transporter (hCRT-BS2M) cDNA clone was isolated from a human brainstem/spinal cord using a PCR and phage plaque hybridization based technique. This clone included an open reading frame of 1,905 base pairs(bp) within a 2,283bp cDNA. Northern blot hybridization detected the expression of corresponding mRNAs most prominently in the skeletal muscle, heart and kidney. Peptide sequence analysis of the hCRT-BS2M protein product revealed 12 putative transmembrane domains. The predicted protein sequence further demonstrates that the hCRT-BS2M has highly conserved amino acid identity with the other members of the sodium dependent plasma membrane transporter family. Transient expression of the hCRT-BS2M in COS-7 cells demonstrates sodium dependent [14C]creatine uptake with a KM value of 14.9 ± 3.0 μM (n=5) that is attenuated by creatine and selective structural analogues of creatine.

Interaction of chronic creatine depletion and muscle unloading: effects on postural locomotor muscles

In some rodent skeletal muscles, hindlimb non-weight-bearing activity induces a shift in the expression of myosin heavy chains (MHCs) that favors the type II isoforms at the expense of type I. Chemically induced chronic creatine depletion results in isomyosin shifts favoring expression of type I MHCs. In this study, creatine depletion was induced separately and in combination with non-weight-bearing activity to determine if the response to lowering this metabolite would counter the MHC transitions expected from non-weight bearing. Creatine depletion was induced by feeding rats a diet supplemented with the creatine analogue beta-guanidinopropionic acid (beta-GPA). Female Sprague-Dawley rats weighing 247 +/- 8 g were randomly assigned to four groups: 1) normal diet control, 2) beta-GPA control (BC), 3) normal diet suspended (NS), and 4) beta-GPA suspended (BS). BC and BS animals were fed a diet containing the creatine analogue for 68 days. Hindlimb non-weight bearing in BS and NS animals was accomplished by tail suspension for the final 30 days of this period. beta-GPA feeding lowered the creatine content of muscles sampled by 65%. Creatine depletion resulted in a 16% increase in citrate synthase activity in the soleus (SOL) and a 24% increase in the plantaris (PLN). In two postural muscles, the SOL and vastus intermedius (VI), tail suspension resulted in large decreases in the type I MHC expression and increases in type IIx and IIb MHCs. In two locomotor muscles, the PLN and medial gastrocnemius, type I MHC declined and type IIb increased with suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP synthesis kinetic properties of mitochondria isolated from the rat extensor digitorum longus muscle depleted of creatine with β-guanidinopropionic acid

A creatine analogue, β-guanidinopropionic acid (β-GPA), was administered in the food (1% w w ) of 8 male rats while 8 control rats received a standard diet. Mitochondrial oxidative capacity and kinetic parameters of mitochondrial ATP synthesis, apparent maximal ATP synthesis rate ( V max) and apparent Michaelis constant for free ADP ( K m), were investigated in the extensor digitorum longus (EDL) muscle. Mitochondrial ATP synthesis rate was measured by a bioluminescent method over a large range of ADP concentration (2–30 μM). As a result of the diet, V max was significantly increased ( P < 0.05) while K m remained unchanged at around 20 μM. Citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase activities were significantly increased (both P < 0.05). V max was tightly correlated with CS activity ( P < 0.001; r = 0.84). It was concluded that the increase in maximal mitochondrial ATP synthesis rate after β-GPA feeding in EDL muscle was essentially due to a general increase in mitochondrial enzyme concentrations.

Metabolic adaptation of skeletal muscles to gravitational unloading

Responses of high-energy phosphates and metabolic properties to hindlimb suspension were studied in adult rats. The relative content of phosphocreatine (PCr) in the calf muscles was significantly higher in rats suspended for 10 days than in age-matched cage controls. The Pi PCr ratio, where Pi is inorganic phosphate, in suspended muscles was less than controls. The absolute weights of soleus and medial gastrocnemius (MG) were approximately 40% less than controls. Although the % fiber distribution in MG was unchanged, the % slow fibers decreased and the % fibers which were classified as both slow and fast was increased in soleus. The activities (per unit weight or protein) of succinate dehydrogenase and lactate dehydrogenase in soleus were unchanged but those of cytochrome oxidase, β-hydroxyacyl CoA dehydrogenase, and citrate synthase were decreased following unloading. None of these enzyme activities in MG changed. However, the total levels of all enzymes in whole muscles decreased by suspension. It is suggested that shift of slow muscle toward fast type by unloading is associated with a decrease in mitochondrial biogenesis. Further, gravitational unloading affected the levels of muscle proteins differently even in the same mitochondrial enzymes. Unloading-related atrophy is prominent in red muscle or slow-twitch fiber 1, 2. Such atrophy is accompanied by a shift of contractile properties toward fast-twitch type 2–9. Further, inhibition of mitochondrial metabolism in these muscles is also reported by some studies 10–14 suggesting a lowered mitochondrial biogenesis, although results from some studies do not necessarily agree 1, 7, 15. However, the precise mechanism responsible for such alterations of muscle properties in response to gravitational unloading is unclear. On the contrary, mitochondrial biogenesis, suggested by mitochondrial enzyme activities and/or mass, is stimulated in muscles with depleted high-energy phosphates by cold exposure 16 and/or by feeding creatine analogue β-guanidinopropionic acid 17–19. Tension production may be inhibited in unloaded antigravity muscles 20, although the muscular activity detected by electromyography is not necessarily decreased 21. Thus, the contents of high-energy phosphates or turnover rate of adenosine triphosphate (ATP), which then affect the mitochondrial energy metabolism, may be altered. Therefore, the responses of high-energy phosphates and metabolic properties of rat hindlimb muscles to gravitational unloading were investigated.

Contractile economy and aerobic recovery metabolism in skeletal muscle adapted to creatine depletion

Mice were treated for 7-12 wk with the creatine analogue beta-guanidinopropionic acid (beta-GPA). Treatment reduced total creatine to approximately 5% of control values in soleus (SOL) and extensor digitorum longus (EDL) muscles. In both muscles from treated mice, phosphorylated beta-GPA accumulated and resting [ATP] decreased by approximately 50%. Relative to controls, cytochrome oxidase and citrate synthase activities increased significantly in EDL from treated mice, but not in SOL; creatine kinase activity decreased significantly in SOL, but not in EDL. Measurements of poststimulation energy metabolism show that the energy cost to maintain tension in SOL and EDL from treated mice was approximately 50% of that in control muscle. Relative to controls, first-order rate constants of poststimulation O2 demand were 2- and 3.6-fold greater in SOL and EDL, respectively, from treated mice. Increased economy of SOL and EDL from treated mice is consistent with previously reported changes in myosin isoenzymes. Increases in rate constants of O2 utilization in creatine-depleted muscle are inconsistent with the hypothesis that cytoplasmic or mitochondrial creatine kinase is rate limiting for cellular respiration.

Actions of the creatine analogue β-guanidinopropionic acid on rat heart mitochondria

The action of the creatine analogue beta-guanidinopropionic acid (beta-GPA) was examined in rat heart mitochondria and in isolated cardiomyocytes or fibres which were permeabilized with the non-ionic detergent saponin to determine the kinetics of mitochondrial creatine kinase for beta-GPA. Fibres and myocytes were subjected to increasing [ADP] in the presence and absence of beta-GPA or creatine, whereas isolated mitochondria received a similar protocol with increasing [ATP]. In isolated mitochondria given ATP, there was a stimulation of respiration by creatine, but no significant stimulation of respiration by beta-GPA. Further studies on fibres from control and beta-GPA-fed rats also found that beta-GPA is not utilized by the mitochondria, as evidenced by a lack of beta-GPA-stimulated respiration (Km for ADP = 142 +/- 23 microM) compared with control (Km for ADP from 161 +/- 23 microM), but no significant change in Vmax. Therefore the rat heart mitochondria are not responsive to beta-GPA as compared with creatine. Interestingly, the fibres from beta-GPA-fed rats had no creatine- or beta-GPA-stimulated respiration (Km for ADP = 57.3 +/- 7.2 microM for control, 54.2 +/- 7.2 microM with creatine, and 53.5 +/- 7.8 microM with beta-GPA). The mitochondria prepared from the hearts of rats exposed for 10 weeks to 1% beta-GPA in their diet had a significant decrease in Vmax. and a significant decrease in Km for ADP. Thus the hearts from beta-GPA-fed animals may be pathologic, due to a disruption of the creatine kinase energy circuit.