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Biomedical subjects

D O Allen

Publications and source records attributed to D O Allen.

At least 19 recordsLinked to original sources
PubMed1988

Modulation of beta-receptors as adult and neonatal cardiac myocytes progress into culture.

Modulation of beta-adrenergic receptors and their ability to respond to beta-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (125I-CYP) was used to identify beta-adrenoceptors on the intact cells. 125I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of beta-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to beta-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional beta-receptors as they progress into culture, and the expression of beta-receptors is not influenced by culture substrates.

Animals↗
PubMed1988

Quantitative differences in the cyclic AMP-lipolysis relationships for isoproterenol and forskolin.

The relationships between cyclic AMP (cAMP) levels and glycerol release (lipolysis) were determined for isoproterenol and forskolin under varied conditions in the isolated fat cell of the rat. Prevention of the inhibitory action of endogenous adenosine [by adenosine deaminase (100 mU/ml) or theophylline (3.3 X 10(-4) M)] resulted in increased levels of cAMP and increased rates of lipolysis with forskolin. However, the relationship between cAMP levels and rates of lipolysis remained the same under all conditions. N6-phenylisopropyladenosine (PIA; an analog of adenosine) abolished the increase in cAMP level produced by isoproterenol (10(-7) M) or forskolin (10(-6) M) and the lipolytic response to forskolin. However, PIA failed to inhibit completely the lipolytic response to isoproterenol. Dose-response curves to isoproterenol were determined in the presence and absence of adenosine deaminase. PIA (10(-6) M) inhibited the increase in cAMP levels under both conditions. PIA also inhibited the lipolytic responses that were associated with increases in cAMP levels, i.e., high concentrations of isoproterenol alone and isoproterenol with adenosine deaminase. A plot of cAMP levels against corresponding rates of lipolysis for all conditions agreed with previous observations that the relationship for isoproterenol differs from that for forskolin. At any concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin. The possibility of a cAMP-independent lipolytic response was discussed.

Adenosine Deaminase↗
PubMed1986

Role of calcium ion in hormone-stimulated lipolysis.

Using the flask-incubated fat cell system, the effects of Ca2+ removal from the incubation medium on the lipolytic system were studied. The removal of Ca2+ resulted in a total abolition of the lipolytic response and the increased cyclic AMP accumulation produced by ACTH. The lipolytic response to isoproterenol and forskolin were reduced approximately 40% by Ca2+ removal, but cyclic AMP accumulation was not altered in the presence of either of these agents using a Ca2+-free medium. The lipolytic response to the dibutyryl analog of cyclic AMP was also reduced by omission of Ca2+ from the incubation medium. It is concluded the Ca2+ is required for the interaction of ACTH with its receptor and the resultant activation of adenylate cyclase. Ca2+ also is required at some step in the lipolytic process distal to cyclic AMP.

Adipose Tissue↗
PubMed1986

Relationships between cyclic AMP levels and lipolysis in fat cells after isoproterenol and forskolin stimulation.

Using the flask-incubated fat cell system, alterations in glycerol release (lipolysis) and cAMP accumulation were determined after incubation with isoproterenol or forskolin. These agents caused concentration-dependent increases in both cAMP accumulation and lipolysis. The maximum responses to forskolin for each variable were greater than the corresponding responses to isoproterenol. The maximum responses to isoproterenol for both cAMP accumulation and glycerol release were increased by the presence of either adenosine deaminase or theophylline. Under these conditions, high concentrations of isoproterenol continued to increase cAMP accumulation while having no further effect on lipolysis. These results support the concept that the maximum response to isoproterenol alone was limited by the accumulation of cAMP within the cells. The maximum response to isoproterenol in the presence of either theophylline or adenosine deaminase (and to forskolin) was limited by some step in the lipolytic process distal to cAMP accumulation. The relationships between cAMP levels and lipolysis for isoproterenol and forskolin were found to be different. A 6-fold increase in cAMP levels was sufficient to maximally increase lipolysis with isoproterenol, whereas the maximum lipolytic response to forskolin was associated with a 20-fold increase in cAMP levels. A plot of log cAMP vs. glycerol release resulted in linear relationships for both drugs. The slope of the line for isoproterenol was significantly greater than that for forskolin. At any given concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin.

Adenosine Deaminase↗
PubMed1985

Rate-limiting steps in isoproterenol and forskolin stimulated lipolysis.

Using the flask-incubated fat cell system, effects of isoproterenol and forskolin on glycerol release, cyclic AMP levels and protein kinase were studied. Isoproterenol increased cyclic AMP levels, protein kinase activity and glycerol release over the same concentration range (10(-9) M to 10(-6) M). Forskolin also increased all three variables in a concentration-dependent manner (10(-7) M to 10(-4) M). The maximum response for each variable was significantly greater with forskolin than with isoproterenol. A combination of isoproterenol and forskolin resulted in an additional increase in cyclic AMP over forskolin alone, but no significant increase in protein kinase activity or glycerol release. These results support the concepts that the maximum lipolytic response to isoproterenol is limited by the accumulation of cyclic AMP and the maximum lipolytic response to forskolin is limited by some step distal to cyclic AMP production, possibly activation of protein kinase. At high concentrations of forskolin or with a combination of forskolin and isoproterenol, cyclic AMP levels were in excess of those needed to maximally activate protein kinase and lipolysis.

Animals↗
PubMed1979

Characterization of the multiple-chamber perifused fat cell system.

A multiple-chamber perifused fat cell system is described. Six chambers containing fat cells were perifused in parallel with buffer. Perifusate was collected for assay of glycerol as an index of lipolytic rates and cells in each chamber can be taken for analysis of biochemical intermediates. The system is so designed that drugs can be infused into the buffer and equally distributed in each chamber or can be individually infused into the buffer to one chamber, allowing for six different conditions to be tested in the same population of fat cells. The time and distribution characteristics of infused material are described. Time relationships are described for isoproterenol and glycerol release and for cyclic AMP levels in the fat cells, and the dose-response relationship between isoproterenol and glycerol release is shown.

Adipose Tissue↗
PubMed1978

Increased cyclic GMP in the end-plate region of denervated frog muscle.

Denervated frog sartorius muscles showed an approximately 2--3 fold increase of cyclic GMP in their end-plate rich regions which did not appear up to 5 weeks after denervation in the normally end-plate-free pelvic region. No increase in cyclic AMP was seen in these preparations. The results suggest that the increase of cyclic GMP is related to processes specific to the region in which end plates are normally present.

Acetylcholine↗
PubMed1977

Appearances of responses to glucagon in cultured neoatal rat heart cells.

Heart cells from neonatal rats have been cultured. The ability of 10(-5)M glucagon to stimulate adenylyl cyclase activity, and to increase the cAMP concentration and the beating rate in these cells was followed as a function of time in culture. The cultured cells show no response to 10(-5)M glucagon until 5 weeks. By contrast, the cells do respond to 10(-5)M epinephrine with an increase in beat rate, adenylyl cyclase activity and cAMP levels when freshly prepared or after 1 week in culture. Previous studies on the newborn rat heart, acutely isolated, have also shown that the neonatal rat heart is insensitive to glucagon until 4-5 weeks after birth. We conclude that the cultured neonatal rat heart cells can also mature in the same time frame with respect to a glucagon response.

Adenylyl Cyclases↗
PubMed1977

Perifused adipose cells, quantitation and kinetics of lipolysis.

The perifused fat cell system is a system with which lipolytic activity can be monitored on a minute-to-minute basis. Thus, the rate at which lipolysis changes following the addition and removal of hormones can be followed. Catecholamines and other lipolytic agents produced a time-dependent increase in lipolysis following addition of agents, and a time-dependent decrease in lipolysis occurred following removal of the agent. ACTH also produced an increase in lipolysis. However, on termination of ACTH infusion, the lipolytic rate did not return to basal level but remained elevated for at least an additional 30 min (persistent phase). The persistent phase could be terminated by removal of Ca2+. Readdition of Ca2+ in the absence of additional ACTH resulted in a rapid increase in glycerol release. No persistant phase occurred following ACTH if the adipocytes were perifused in a Ca2+-free buffer. However, if Ca2+ was added to the system 20 min after termination of ACTH infusion, lipolysis increased to a rate greater than that obtained initially by infusing ACTH in a Ca2+-free buffer. It is concluded that ACTH is bound to some component of the fat cell in a Ca2+ independent, tenacious manner, and the full manifestation of that binding is dependent on the presence of Ca2+.

Adipose Tissue↗
PubMed1976

On the lipolytic action of parathyroid hormone in man.

An investigation was carried out to determine whether bovine PTH stimulates lipolysis in human fat tissue, whether this action is mediated by cyclic adenosine 3', 5'-monophosphate and whether the N-terminal 1-34 peptide of bovine PTH is responsible for the lipolytic effect. Studies were also performed to determine if parathyroid extract (PTE) produces lipolysis in normal subjects and in patients with pseudohypoparathyroidism in whom there is a defect in the adenylate system in response to PTH in the renal cortex and presumably in the skeletal system as well. It was found that highly purified bovine PTH in the concentration range between 10(-9) M and 10(-5) M stimulated lipolysis in vitro by human fat in a dose-dependent manner. Significant increases in glycerol production were observed at concentrations of PTH as low as 10(-9) M and maximal increases were seen at 10(-6) M. The hormone significantly increased the concentration of cyclic adenosine 3' ,5'-monophosphate in fat tissue. The synthetic N-terminal 1-34 peptide of bovine PTH was as effective as the native hormone in stimulating glycerol production at a concentration of 10(-9) M-10(-6) M. PTE, 100 mU per kg per min for 30 min given intravenously, produced transient increases in the concentration of plasma free fatty acid in each of eight normal subjects, three patients with hypoparathyroidism and eight patients with pseudohypoparathyroidism. Purified bovine PTH also increased plasma free fatty acid in each of two normal subjects. It is concluded that PTH stimulates lipolysis in human subcutaneous fat, that this action of the hormone is mediated through cyclic adenosine 3', 5'-monophosphate and that the N-terminal 1-34 peptide portion of the hormone is responsible for this lipolytic action. Further, PTE stimulates lipolysis in vivo in man. There appears to be no defect in the adenylate cyclase system in the fat cell in response to PTH in patients with pseudohypoparathyroidism.

Adipose Tissue↗
PubMed1976

Demonstration that cyclic adenosine 3',5'-monophosphate mediates the lipolytic action of parathyroid hormone.

Studies were carried out with rat epididymal fat pads first to compare the effects of the synthetic N-terminal 1-34 peptide of bovine parathyroid hormone and of the native hormone to determine whether this portion of the molecule is responsible for the lipolytic action of the hormone and second to determine whether this biologic action of parathyroid hormone is mediated by cyclic adenosine 3',5'-monophosphate. The N-terminal polypeptide was as effective as the native hormone in stimulating lipolysis in the concentration range between 10(-8) M and 10(-6) M. Parathyroid hormone stimulated lipolysis by isolated fat cells. The concentration of cyclic adenosine 3',5'-monophosphate in the fat pads was significantly increased by the hormone (10(-6)M). Lipolytic stimulation by parathyroid hormone (10(-6)M) was diminished by insulin (100 muU/ml) and prostaglandin E1 (1 mug/ml), both of which are known inhibitors of lipolysis. The findings indicate that the amino-terminal 1-34 peptide portion of parathyroid hormone is responsible for the lipolytic action and that this effect is mediated through cyclic adenosine 3',5'-monophosphate.

Adipose Tissue↗
PubMed1976

Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.

Adenylyl Cyclases↗
PubMed1976

Effect of acetylcholine on glycogen phosphorylase activity and cyclic nucleotide content in isolated perfused rat hearts.

Acetylcholine (1muM) increased cyclid GMP content in paced perfused rat hearts within 15 sec., with peak content occurring at 1 min. No effect of acetylcholine on cyclic AMP content, phosphorylase activity or glycogen synthase was observed. Epinephrine (1muM) infusion increased both cyclic AMP content and phosphorylase, but did not alter cyclic GMP content or glycogen synthase activity. When acetylcholine was infused during the second min. of a 2 min. infusion of epinephrine, the cholinergic agent increased cyclic GMP and reduced the stimulated phosphorylase activity and elevated cyclic AMP.

Acetylcholine↗