Effects of insulin on glucose metabolism in isolated heart myocytes from adult rats

The study examined the effect of insulin on glucose metabolism in freshly isolated calcium-tolerant heart myocytes from adult rats. The uptake of 2-deoxyglucose demonstrated an initial lag in response to insulin and the maximal insulin effect was not attained until after 3 min preincubation with the hormone. A dose-response study of 14CO2 production from [14C]glucose revealed that the maximum insulin stimulation of glucose utilization occurred with 5 mU/ml. Both the uptake and the oxidation of glucose proceeded at a linear rate in the absence and presence of insulin. However, insulin exerted a greater effect on the uptake (42-54%) than on the oxidation (17-22%) of exogenous glucose. Incorporation of glucose into glycogen was markedly increased by insulin and resulted in the myocyte glycogen concentration returning to in vivo levels. In the absence of insulin, glucose incorporation plateaued within 10 min of incubation and the glycogen concentration was not altered. Our findings also indicate that at equilibrium, insulin-treated cells exhibited a higher glycogen turnover rate. It thus appears that insulin exerts a differential effect on the different pathways in glucose metabolism in the isolated cardiac cells. This may be related in part to their quiescent state and lower energy demand.     

A method to collect isolated myocytes and whole tissue from the same heart

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and -amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs.-Autoradiographic studies carried out in the presence or absence of the toxin -amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in -amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

In vitro transcription of calliphorin genes in isolated nuclei of fat body from larvae of Calliphora vicina

Nuclei from fat body of different developmental stages of Calliphora vicina were isolated. They appear to be polyploid and show polytene chromosomes. The isolated nuclei were incubated with [³²P]GTP and the RNA transcribed in vitro was hybridized with a DNA fragment encoding a polypeptide subunit of calliphorin. The isolated nuclei transcribe the calliphorin-mRNA correctly and with the same stage specificity as observed in vivo.     

In vitro synthesis and stability of RNA in isolated nuclei from bovine lymphocytes

Nuclei from Concanavalin A-stimulated lymphocytes (30 hr after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14–20% of the $\text{in vitro}$ labeled RNA is polyadenylated. Poly(A) (?)RNA from both types of nuclei sediments from 4–5S up to more than 30S on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18S than nuclei from resting cells. The same holds for poly(A) (+)RNA. Poly(A) (?) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions poly(A) (+)RNA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells.     

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.