Adenine nucleotide synthesis de novo in mature rat cardiac myocytes.

Inadequate oxygenation of cardiac muscle leads to rapid loss of high energy compounds essential for contractile function. ATP can be regenerated by synthesis de novo, a route operating at a relatively slow rate in the heart. Myocytes isolated from mature rat heart have been used to measure the rate of ATP synthesis de novo from both [14C]glycine and [14C]ribose. Incorporation of glycine into ATP is accelerated 10-fold in the presence of 1 mM ribose. Myocytes also accumulate both precursors into IMP and four other metabolites on the de novo synthesis pathway. These metabolites represent 80% of the glycine entering the pathway. The potential of de novo synthesis for restoration of adenine nucleotides appears to be limited by the rates of early reactions, adenylosuccinate synthetase being only one of the enzymes operating at a sufficiently slow rate to make this pathway an inherently weak route for the restoration of normal energy status in post-ischemic myocardium. Interventions are being sought to alleviate these apparent metabolic delays.

Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes.

Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7-8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine.

5'-Nucleotidase activity of isolated mature rat cardiac myocytes.

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart.

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.

Calcium antagonists and adenine nucleotide metabolism in rat heart.

Attempts to identify mechanisms by which calcium antagonists might influence intracellular metabolism have not yet yielded conclusive findings. In this study bepridil, verapamil, nifedipine, and nisoldipine were found to have no influence on the rate of rat heart myosin adenosine triphosphatase or the calcium dependence of myofibrillar adenosine triphosphatase. None of these calcium antagonists alters the rate of reaction of any of the adenine nucleotide catabolic or adenosine salvage enzymes, adenylate kinase, creatine kinase, adenosine kinase, adenosine deaminase, or 5' nucleotidase, in extracts of rat heart. All four compounds, however, reduced, apparently in a non-specific manner, the rate of uptake of adenosine by myocytes isolated from rat heart. It is concluded that calcium antagonists may, through intercalation with the sarcolemmal membrane, inhibit efflux of adenosine formed by catabolism of adenine nucleotides in ischaemic myocytes. This might offer therapeutic advantage since the intracellular concentration of adenosine would thereby be increased, allowing an increased rate of incorporation of adenosine into the adenosine triphosphate pool in reoxygenated myocardium.

Salvage mechanisms for regeneration of adenosine triphosphate in rat cardiac myocytes.

Since mitochondrial oxidative phosphorylation does not produce sufficient adenosine triphosphate for rapid restoration of contractile function in myocardium reoxygenated after ischaemia alternative non-oxidative routes by which purine nucleosides or bases may be used to increase the cytoplasmic adenine nucleotide pool were studied. Comparative rates of uptake and salvage of adenosine, adenine, and hypoxanthine were determined using myocytes isolated from adult rat heart. For each precursor reactions limiting the overall rate at which substrate in the extracellular fluid is incorporated into the intracellular adenosine triphosphate pool were identified. Adenosine was salvaged at twice the rate seen with adenine in the presence of ribose, whereas the rate of salvage of hypoxanthine, in the presence of ribose, was only 5% of that for adenosine. Adenine may be an advantageous substrate since high concentrations of adenine are not inhibitory to salvage and do not influence cardiac haemodynamics. Salvage of hypoxanthine appeared to be limited by the rate of adenylosuccinate synthetase, which was present at less than 1% of the adenylosuccinase rate in rat, rabbit, and beef heart. In addition, since salvage of bases is dependent on the availability of phosphoribosylpyrophosphate rates of synthesis of phosphoribosylpyrophosphate were measured in whole myocytes from ribose and in cytoplasmic extracts from ribose and from ribose-5-phosphate. Metabolic sites were identified at which interventions designed to accelerate salvage rates might best be directed.

Syphilis in pregnancy: experience from a rural aboriginal community.

Syphilis in pregnancy is seldom encountered in urban Australia. This retrospective study reports on the high prevalence of syphilis and its associated complications in a group of pregnant Aboriginal women attending the Dareton Community Health Centre for antenatal care between September, 1989 and September, 1991. During this time, the centre provided antenatal care for 71 Aboriginal pregnancies and maternal syphilis was found in 20 (28%). The mean age of the women was 21.3 years (range of 14-33 years). The perinatal mortality rate was 48 per 1,000 births. All stillbirths were associated with maternal syphilis. The relative risk of stillbirth in the presence of syphilis was 4 (95% CI 2.56-6.25, p = 0.022). Prematurity was associated with maternal syphilis. The odds ratio for premature delivery was 21.5 (95% CI 2.26-2.05, p = 0.003). There was no statistical difference between the mean birth-weight and mean placental weight of term infants born to women with syphilis and women without syphilis (p = 0.35 and p = 0.31 respectively). When the placental:birth-weight ratios were considered, an apparent larger placenta was seen in association with syphilis but this was secondary to the lower birth-weights in association with prematurity. Late and poor attendance for antenatal care was a feature of all women attending this centre and not a feature associated with syphilis. The treatment and follow-up of syphilis were hindered by poor compliance.

Variation in receptor status between primary and metastatic breast cancer.

Steroid hormone receptors for oestradiol (ER) and progesterone (PR) have been measured sequentially in primary and metastatic breast cancer to determine whether receptor status remains concordant in individual patients. Serial receptor measurements from the same tumor site showed a change from receptor-positive to receptor-negative tissue with time in some patients. Receptors were measured in primary tumor and later metastatic disease or tumor of the contralateral breast in 28 patients. Concordant results were found in 46% of cases. Six of nine patients with ER-positive primary tumors developed ER-negative metastases and four of five PR-positive primary tumors were associated with PR-negative secondary deposits. Nine of 19 patients with ER-negative primary tumors later had ER-positive metastases, and three of 15 PR-negative primary tumors were associated with PR-positive secondary deposits. Possible false-negative ER measurements (ER-, PR+) in primary tumors, and interval treatment with chemotherapy or hormonal manipulation between biopsies may have contributed to these changes in receptor status. Six patients changing from receptor-negative primary tumors to receptor-positive metastases underwent hormonal therapy of metastatic disease, and four had objective responses to treatment. Care should be taken when using receptor measurements from primary breast tumors to help decide treatment for later metastatic disease.