Biomedical subjects

G F Merrill

Publications and source records attributed to G F Merrill.

At least 37 records · Page 2Linked to original sources

PubMed1991

Route dependent effects of 2-chloroadenosine and theophylline in isolated perfused guinea pig hearts.

STUDY OBJECTIVE: The adenosine hypothesis for the metabolic regulation of coronary blood flow remains controversial, in part because of differences in results obtained between intravascular and endogenously released adenosine. The main objective of this study was to compare the responses of coronary perfusate flow to intravascular and pericardial 2-chloroadenosine and theophylline. The periocardial route was used to simulate physiological conditions. DESIGN: An isolated, perfused guinea pig heart preparation was used. Hearts were submerged in 10 ml Krebs-Ringer bicarbonate solution having the same chemical composition as that used to perfuse the coronary vasculature. The submersion fluid was assumed to simulate myocardial interstitial fluid. Hearts were divided into four experimental groups. The first group (n = 6) was used to check that the submerged hearts functioned normally. The second group (n = 12) was used to compare differences between intravascular and pericardial 2-chloroadenosine. The third group (n = 16) was used to evaluate the two routes of administration of 2-chloroadenosine on transmural distribution of coronary flow. The fourth group (n = 24) was used to test the effects of hypoxia in the absence and presence of pericardial theophylline. EXPERIMENTAL MATERIAL: Hearts were obtained from adult guinea pigs of either sex weighing 300-400 g. MEASUREMENTS AND MAIN RESULTS: All submerged hearts were functionally stable for at least 30 min. Pericardial 2-chloroadenosine was about 1000 times less potent than intravascular 2-chloroadenosine in relaxing the coronary vasculature. Venous concentrations of pericardial 2-chloroadenosine were significantly greater than those of intravascular 2-chloroadenosine, at 0.72(SEM 0.17) v 0.18(0.02) mumol.litre-1. Pericardial 2-chloroadenosine caused more uniform distribution of radiotracer microspheres than intravascular 2-chloroadenosine. Finally, pericardial theophylline significantly attentuated the vasodilator response to hypoxia but not to exogenous adenosine. CONCLUSION: The isolated submerged heart is a viable preparation for investigating the adenosine hypothesis. Our results suggest that intravascular adenosine does not simulate physiological conditions of interstitial adenosine release and action.

2-Chloroadenosine↗

PubMed1991

Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line.

Dihydrofolate reductase (DHFR) enzyme is preferentially synthesized in proliferative cells. A mouse muscle cell line resistant to 300 microM methotrexate was developed to investigate the molecular levels at which DHFR is down-regulated during myogenic withdrawal from the cell cycle. H- alpha R300T cells contained 540 copies of the endogenous DHFR gene and overexpressed DHFR mRNA and DHFR protein. Despite DHFR gene amplification, the cells remained diploid. As H- alpha R300T myoblasts withdrew from the cell cycle and committed to terminal differentiation, DHFR mRNA levels and DHFR synthesis rates decreased with closely matched kinetics. After 15 to 24 h, committed cells contained 5% the proliferative level of DHFR mRNA (80 molecules per committed cell) and synthesized DHFR protein at 6% the proliferative rate. At no point during the commitment process did the decrease in DHFR synthesis rate exceed the decrease in DHFR message. The decrease in DHFR mRNA levels during commitment was sufficient to account fully for the decrease in rates of DHFR synthesis. Furthermore, DHFR mRNA remained polysomal, and the average number of ribosomes per message remained constant (five to six ribosomes per DHFR mRNA). The constancy of polysome size, along with the uniform rate of DHFR synthesis per message, indicated that DHFR mRNA was efficiently translated in postreplicative cells. The results support a model wherein replication-dependent changes in DHFR synthesis rates are determined exclusively by changes in DHFR mRNA levels.

Animals↗

PubMed1991

Adenosine deaminase and adenosine attenuate ventricular arrhythmias caused by norepinephrine.

Twenty-five beagles weighing 9.1 +/- 0.4 kg were used to investigate the arrhythmogenic effects of divided doses of intracoronary norepinephrine (50-200 ng.kg-1.min-1) in the absence and the presence of adenosine deaminase (5 U.kg-1.min-1). A dose of norepinephrine (100 ng.kg-1.min-1) that caused 66 +/- 17% ectopy in the absence of adenosine deaminase caused only 16 +/- 14% ectopy (P less than 0.05) in the presence of the enzyme. Ventricular tachycardia caused by 200 ng.kg-1.min-1 norepinephrine was reduced from 1.2 +/- 0.3 to 0.1 +/- 0.1 bouts/10 cardiac cycles (P less than 0.05) by adenosine deaminase. In five additional dogs, intracoronary adenosine (0.11 mumol/min) terminated sustained norepinephrine-induced (200 ng.kg-1.min-1) ventricular tachycardia within 23 +/- 9 s (P less than 0.05). As long as the adenosine infusion was maintained, a normal sinus rhythm was observed. We conclude that both adenosine and adenosine deaminase significantly attenuate norepinephrine-induced ventricular arrhythmias. A common element beyond the deamination of adenosine, quite possibly ammonia, appears to account for these results.

Adenosine↗

PubMed1990

An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression.

The gene encoding dihydrofolate reductase (DHFR) is down-regulated as myoblasts withdraw from the cell cycle and commit to terminal differentiation. To localize cis-acting elements involved in regulating DHFR gene expression, the DHFR promoter and upstream region, together with differing amounts of contiguous intragenic sequence, were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The resulting fusion genes were stably transformed into muscle cells, and CAT mRNA levels were measured in proliferative myoblasts and committed myocytes. A gene consisting of the -850/+465 region of DHFR (numbers refer to distance in base pairs from transcription initiation site) fused to CAT was efficiently expressed in proliferating myoblasts and was appropriately down-regulated during commitment. A gene consisting of the -850/+60 region of DHFR fused to CAT was poorly expressed in proliferating myoblasts and was not down-regulated during commitment. When inserted between the Rous sarcoma virus promoter and CAT sequence of RSVpCAT, the +61/+465 region of the DHFR gene augmented CAT mRNA expression in muscle cell transformants but did not confer a regulated pattern of expression. Our data indicate that DHFR sequences between +60 and +465 are required but are not sufficient for replication-dependent expression. The DHFR sequences may be operating at either a transcriptional or posttranscriptional level.

Animals↗

PubMed1990

Plasma lipid concentrations in college students performing self-selected exercise.

Plasma cholesterol and other blood lipids were determined in 64 college juniors and seniors during January and February 1989 (before exercise) and again during April and May 1989 (after exercise). A 14-week period of self-selected exercise was interposed between the before and after exercise cholesterol determinations. Cholesterol concentrations ranged from 127-273 mg/dl (before exercise) to 131-261 mg/dl (after exercise) in this group of students who were 22 +/- 1 years of age. There were no statistically significant differences in cholesterol concentrations between the genders. Students could be further subdivided into groups with low (144 +/- 3 mg/dl, n = 12), medium (176 +/- 6 mg/dl, n = 35), and high (224 +/- 6 mg/dl, n = 17) concentrations of cholesterol. Only students in the high cholesterol group experienced a significant reduction in total cholesterol and low-density-lipoprotein cholesterol after 14 weeks of exercise. High-density-lipoprotein cholesterol concentrations were significantly higher in women than in men in both the medium and high cholesterol subgroups before and after exercise. From these results it appears that a significant fraction of the young adult college population could be at increased risk of coronary heart disease because of inappropriately high concentrations of cholesterol. Moreover, self-selected exercise, if engaged in regularly, can reduce blood cholesterol significantly in students with high cholesterol concentrations.

Adult↗

PubMed1990

Positive inotropic effect of carcinine in the isolated perfused guinea pig heart.

Carcinine (beta-alanylhistamine) is a recently discovered compound that is present in the hearts of several mammalian species, including man. Although the function of carcinine is unknown, its structural similarity to histamine, a compound known to have profound effects on the mammalian heart, and to carnosine (beta-alanylhistidine), a compound which we have previously shown to serve as a histamine source, led to the hypothesis that carcinine may play a role in mammalian cardiac physiology. We therefore administered several doses of carcinine (10, 25, 50, 75, and 100 micrograms) to isolated, perfused guinea pig hearts in a Langendorff apparatus. Carcinine exerted a dose-dependent positive inotropic effect, similar to that of histamine. Comparable doses of carnosine yielded no measurable change in contractility. We conclude that carcinine appears to be a positive inotrope in the mammalian heart, and may play a role in cardiac physiology via its metabolic link to histamine.

Animals↗

PubMed1990

Role of adenosine in catecholamine-induced global coronary functional hyperemia in isolated guinea pig hearts.

This study was designed to test the hypothesis that endogenous adenosine participates in the global coronary functional hyperemia accompanying intracoronary infusions of norepinephrine (NE) and isoproterenol (ISO). Intracoronary adenosine deaminase (ADA) was employed to test the hypothesis in isolated, perfused guinea pig hearts. We measured coronary perfusate flow (CPF) at a constant coronary perfusion pressure. Heart rate (HR), left ventricular pressure, and its rate of development were also measured. Global myocardial oxygen consumption (MVO2) and oxygen extraction were calculated, and blood gases and pH were measured routinely in inflow and outflow perfusate samples. In the absence of ADA, NE and ISO increased HR 67 +/- 6 and 106 +/- 11 beats.min-1, left ventricular pressure development 519 +/- 46 and 375 +/- 35 mm Hg.s-1, MVO2 22 +/- 2 and 28 +/- 3 microliters.min-1.g-1, and CPF 1.6 +/- 0.2 and 2.2 +/- 0.2 ml.min-1.g-1, respectively. With constant infusion of ADA (4.5 U.min-1.g-1, a dose which produces no direct effects on cardiac function) for 4 min, similar increments in HR and left ventricular pressure development were achieved with both catecholamines. Corresponding changes in MVO2 (9 +/- 2 and 6 +/- 3 microliters.min-1.g-1) were significantly less than those seen in the absence of ADA. Moreover, CPF did not increase in response to NE and ISO in the presence of ADA. These findings support an important role for adenosine in catecholamine-induced global coronary functional hyperemia in isolated, perfused guinea pig hearts.

Adenosine↗

PubMed1990

Coronary vasodilation caused by intravenous cocaine in the anesthetized beagle.

The aim of this study was to determine the effect of intravenous cocaine on the coronary circulation in the dog. Sixteen beagles separated into three groups were administered either cocaine (n = 8) or lidocaine (n = 4) at doses of 0.4, 2.0, and 10.0 mg/kg under conditions of constant coronary blood flow. A third group of beagles (n = 4) was administered cocaine under conditions of natural coronary blood flow. In the first group, the lowest dose of cocaine had no significant effect on coronary perfusion pressure, even though it increased mean systemic arterial pressure by 10% (p less than 0.05). The second two doses decreased coronary perfusion pressure by 13 (p less than 0.05) and 68% (p less than 0.05), respectively. In the second group, the lowest dose of lidocaine did not significantly affect coronary perfusion pressure. However, the second two doses significantly decreased coronary perfusion pressure by 22 (p less than 0.05) and 45% (p less than 0.05), respectively. Under conditions of natural coronary blood flow and coronary perfusion pressure, these same doses of cocaine increased coronary blood flow by 25, 63, and 175%, respectively. All coronary vascular responses occurred 60 s after administration of cocaine or lidocaine. We conclude that cocaine causes rapid, dose-dependent coronary vasodilation in the anesthetized beagle. The coronary vasodilation appears to be related to cocaine's known, local anesthetic properties.

Anesthesia↗

PubMed1989

Transcriptional repression of the mouse dihydrofolate reductase gene during muscle cell commitment.

A differentiation-competent mouse muscle cell line containing 50-100-times the diploid number of dihydrofolate reductase (DHFR) genes was used to study regulation of DHFR mRNA levels during myogenic withdrawal from the cell cycle. Quantitative RNase protection assays showed DHFR mRNA levels decreased 15-fold during commitment; DHFR pre-mRNA levels decreased 7-fold. Concomitantly, transcription products were analyzed by hybridization to Southern blots of dhfr-containing plasmids. Control run-on assays performed on nonamplified parental cells indicated that run-on signals measured in amplified cells were dhfr amplicon-specific. Run-on signals were sensitive to alpha-amanitin, indicating RNA polymerase 2 specificity, and did not hybridize to pBR322 sequences, demonstrating hybridization stringency. Comparison of run-on signals hybridizing to DNA fragments representing either the 5' end of the gene or the entire gene showed that transcriptional repression occurred within the first 660 bases of the 30-kilobase gene, consistent with regulation at the level of either initiation or early pretermination. In contrast to the DHFR gene, DNA 5' to all but the first few bases of the DHFR coding region (between -1000 and +60 base pairs from the preferred cap site) showed strong run-on transcription in both proliferative and committed cells. Northern blot analysis using a probe complementary both to the dhfr coding region and the upstream region showed a uniform decrease in all detectable transcripts. No commitment-dependent changes in dhfr cap site usage, splicing, or polyadenylylation site usage were detected. Our results support a transcriptional model for regulation of DHFR mRNA levels.

Animals↗

PubMed1989

Maintenance of dihydrofolate reductase enzyme after disappearance of DHFR mRNA during muscle cell differentiation.

Terminally differentiating mouse muscle cells were used to examine the relationship between myogenic withdrawal from the cell cycle and the levels of dihydrofolate reductase (DHFR) mRNA and DHFR activity. Differentiation was induced by removal of fibroblast growth factor activity from the medium. DHFR mRNA was measured by a RNase protection assay. DHFR activity was measured by a spectrophotometric assay and by a [3H]methotrexate binding assay. Proliferative myoblasts contained four DHFR mRNA molecules and 1.8 X 10(5) DHFR enzyme molecules. By 12.5 h after induction, when [3H]thymidine labeling indices showed all cells had withdrawn from the cell cycle, DHFR mRNA levels had declined to 0.7 copies per cell. In contrast, myogenic withdrawal did not result in reduced DHFR activity. Qualitatively similar results, i.e. down-regulation of mRNA and constitutive expression of activity, were observed in a methotrexate-selected muscle cell line with greater than 50-fold amplification of the DHFR gene. Enzyme synthesis rate and stability measurements indicated that persistence of DHFR activity in postreplicative cells was due to a long enzyme lifetime rather than to continued synthesis from residual normal DHFR mRNA or an alternative mRNA species not detected by the RNase protection assay. Unlike DHFR, thymidine kinase (TK) activity disappeared rapidly as muscle cells differentiated. Both DHFR mRNA and TK mRNA are expressed in a replication-dependent manner; however, the enzymes encoded by these messages are subject to different fates in postreplicative cells.

Animals↗

PubMed1989

Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes.

A muscle cell strain capable of forming contracting myotubes was isolated from an established rat embryo cell line. The myogenic cells, termed rat myoblast omega or RMo cells, have a diploid complement of chromosomes (n = 42). In the presence of mitogen-containing growth medium, RMo cells proliferated with a cell generation time of about 12 hours. In mitogen-depleted medium, RMo cells withdrew from the cell cycle and formed myotubes that spontaneously contracted. Differentiated RMo cells produced creatine kinase isozymes in a ratio characteristic of skeletal muscle cells. RMo cells were easy to cultivate. Cells proliferated and differentiated equally well on gelatin-coated or noncoated culture dishes, at clonal or mass culture densities, and in all basal media tested. In most experiments, growth medium consisted of horse serum-containing medium supplemented with either chicken embryo extract or FGF activity; cells proliferated equally well in medium containing unsupplemented calf serum. RMo cells differentiated if growth medium was not replenished regularly. Alternatively, differentiation was induceable by incubation in mitogen-depleted medium consisting of basal medium supplemented either with 10(-6) M insulin, 0.5% serum, or 50% conditioned growth medium. RMo cells were competently transformed with cloned exogenous genes. Because it forms functional myofibrils, the RMo cell line constitutes a useful model system for studying the cell biology and biochemistry of proteins involved in contractile apparatus assembly and muscle disease.

Animals↗

PubMed1989

Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA.

The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.

Animals↗

PubMed1989

Canine coronary vasodepressor responses to hypoxia are abolished by 8-phenyltheophylline.

Anesthetized randomsource mongrel dogs of either sex were instrumented to investigate the effects of 8-phenyltheophylline on changes in coronary perfusion pressure caused by systemic hypoxia under conditions of controlled constant coronary blood flow. In the absence of 8-phenyltheophylline, coronary perfusion pressure decreased from 98 +/- 10 to 69 +/- 4 mmHg (P less than 0.05) at the end of 3 min of systemic hypoxia [arterial partial pressure of oxygen (PO2) = 23 +/- 2 mmHg]. Calculated coronary vascular resistance decreased concomitantly by 30 +/- 5% (P less than 0.05). In the presence of continuously infused 8-phenyltheophylline, equally severe hypoxia increased coronary perfusion pressure from 112 +/- 10 to 129 +/- 13 mmHg (P less than 0.05). Under these conditions, calculated coronary vascular resistance increased 14 +/- 3% (P less than 0.05). Dose-dependent attenuation of the coronary vasodilator response to exogenous adenosine under normoxic conditions was produced by 8-phenyltheophylline. In vehicle-treated dogs, repeat bolus injections of adenosine consistently lowered coronary perfusion pressure by 45 +/- 15%. The vasodepressor response did not vary from one injection to the next. These data demonstrate that under conditions of controlled constant coronary blood flow, treatment with 8-phenytheophylline abolishes coronary vasodilation caused by systemic hypoxia.

Adenosine↗

PubMed1988

Regulation of thymidine kinase protein levels during myogenic withdrawal from the cell cycle is independent of mRNA regulation.

Replication-dependent changes in levels of enzymes involved in DNA precursor biosynthesis are accompanied frequently by changes in levels of cognate mRNA. We tested the common assumption that changes in mRNA levels are responsible for growth-dependent expression of these enzymes using a line of mouse muscle cells that irreversibly withdraws from the cell cycle as part of its terminal differentiation program. Thymidine kinase (TK) mRNA, activity, and protein levels were quantitated in cells transformed with multiple copies of the chicken TK gene. The decline in TK mRNA (both whole cell and cytoplasmic) during myogenesis was poor (2-fold average) and variable (1.2 to 8-fold). In contrast, TK activity always was regulated efficiently (20-fold), even in cells which regulated TK mRNA very poorly. Thus, regulation of TK activity was independent of TK mRNA regulation as myoblasts withdrew from the cell cycle. A TK/beta-galactosidase fusion protein was used to derive an antibody against chicken TK. Immunoblot and immunoprecipitation analyses demonstrated TK protein levels, like TK activity levels, declined to a greater extent than TK mRNA levels. Thus, TK activity likely was regulated by a mechanism involving either decreased translation of TK mRNA or increased degradation of TK protein in committed muscle cells.

Animals↗

PubMed1988

Adenosine deaminase attenuates canine coronary vasodilatation during regional non-ischaemic myocardial hypoxia.

To test the hypothesis that adenosine contributes to the coronary hyperaemia produced by regional non-ischaemic myocardial hypoxia coronary blood flow and myocardial oxygen extraction and consumption were continuously monitored in 21 anaesthetised open chest dogs under the following conditions: control 1--postinstrumentation, steady state control; hypoxia 1--3-5 min of regional (LAD) hypoxaemia (partial pressure of oxygen, PO2, 21.4(2.0) mmHg (3.1(0.2) kPa), coronary arterial oxygen content, CaO2, 3.9(0.4) ml.100 ml-1 (39(4) ml.litre-1): control 2--repeat steady state control; and hypoxia 2--3-5 min repeat regional hypoxaemia (PO2 18.9(2.4) mmHg (2.5(0.3) kPa); CaO2 3.6(0.6) ml.100 ml-1 (36(6) ml.litre-1) blood). Left anterior descending artery perfusion pressure was held constant for all conditions. Control 2 and hypoxia 2 were performed in the presence of locally infused adenosine deaminase (n = 16) or saline vehicle (n = 5). The 16 dogs given adenosine deaminase were further subdivided into those perfused with blood deoxygenated by a donor canine lung (group 1, n = 11) and those perfused with blood from a paediatric oxygenator (group 2, n = 5). Systemic haemodynamics, heart rate, and coronary arterial PO2 and O2 contents were similar during the two control periods and during the two exposures to hypoxia in all three groups. Left anterior descending artery blood flow increased by approximately 400% (p less than 0.05) in all three groups during the first exposure to hypoxia. Myocardial oxygen consumption was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1988

Adenosine deaminase attenuates norepinephrine-induced coronary functional hyperemia.

Responses to norepinephrine (NE) before and after treatment with adenosine deaminase (ADA) were examined in anesthetized dogs. In four dogs repeatable changes in coronary blood flow, myocardial oxygen extraction and consumption, left ventricular +dP/dtmax, and heart rate (HR) were demonstrated during two successive intracoronary infusions of 0.13 micrograms.kg-1.min-1 NE. In eight dogs, the NE-induced hyperemia was decreased from +150 to +67%, the change in myocardial oxygen consumption (MVo2) was attenuated from +177 to +101% by ADA, and the increase in HR was reduced from +28 to +16%. In six dogs, the increase in HR caused by NE before ADA was maintained after ADA by atrial pacing. The NE-induced hyperemia and the increase in MVo2 were again decreased by ADA. Similar results were observed in 12 other dogs with hearts paced at a constant, elevated rate during control as well as during both infusions of NE. In all groups, the O2 extraction response to increased MVo2 increased and the flow response decreased after ADA. In six dogs nitroprusside was infused during NE after ADA. When coronary flow was restored to the same level observed before deaminase, MVo2 was not diminished. These results support a role for adenosine in the coronary functional hyperemia accompanying NE activation of the canine myocardium.

Adenosine↗

PubMed1988

Coronary vasodilation during global myocardial hypoxia: effects of adenosine deaminase.

Isolated, perfused guinea pig hearts were infused with intracoronary adenosine deaminase to investigate the contribution of endogenous adenosine to the coronary vasodilation of global myocardial hypoxia. Coronary perfusate pressure was held constant at 70 cmH2O throughout the experiment. We measured retrograde aortic inflow (assumed to equal total antegrade coronary flow) for 3-5 min of hypoxia before and 4 min after initiation of intracoronary adenosine deaminase infusion (4 U.g-1.min-1). In the absence of adenosine deaminase mild global hypoxia increased coronary perfusate flow 60%. In the presence of adenosine deaminase the response was limited to a 5% increment. Myocardial O2 consumption was significantly reduced during hypoxia in the presence of adenosine deaminase. In a second group of hearts, moderate global hypoxia increased coronary perfusate flow 125%. This was limited to a 53% increment in the presence of adenosine deaminase. Adenosine deaminase vehicle had no measurable effect on coronary perfusate flow responses to repeat mild hypoxia in a third group of hearts. We conclude that endogenous adenosine is singularly important in the coronary vasodilation of mild global myocardial hypoxia, but that other regulatory mechanisms might also contribute during moderate hypoxia.

Adenosine Deaminase↗

PubMed1987

The chicken thymidine kinase gene is transcriptionally repressed during terminal differentiation: the associated decline in TK mRNA cannot account fully for the disappearance of TK enzyme activity.

Thymidine kinase (TK) is representative of a class of enzymes involved in DNA precursor biosynthesis that declines as cells withdraw from the cell cycle. If TK activity is regulated exclusively by the availability of messenger RNA, changes in enzyme activity levels should not precede or excede changes in TK mRNA levels. This prediction was tested in several tissues during chicken embryogenesis and in differentiating muscle cells in culture. A sensitive method of determining absolute TK mRNA levels was developed. A synthetic complimentary RNA probe spanning an intron acceptor site in the chicken TK gene was hybridized with cellular RNA or synthetic colinear TK RNA of known concentration. After RNase digestion and gel electrophoresis, the intensity of the protected fragment was used to calculate absolute TK mRNA levels. As few as 0.02 molecules of TK mRNA per cell could be measured accurately. Depending on the tissue type, 8-day embryos contained between 3 and 12 TK mRNAs per cell. Proliferating mouse muscle cells transformed with the chicken TK gene contained between 30 and 150 TK mRNAs per cell. Both in vivo and in vitro, TK mRNA levels declined as cells withdrew from the cell cycle during differentiation. In vivo, the decline in TK activity never preceded or exceeded observed changes in TK mRNA. However, in the cell culture system, TK activity consistently declined to a greater extent than TK mRNA. Thus, a translational or a post-translational mechanism must also be operative in controlling TK activity levels. Estimation of transcription rates in nuclei isolated from proliferating and differentiated muscle cell transformants indicated that the TK gene was transcriptionally repressed in postreplicative cells.

Animals↗