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Biomedical subjects

L T Potter

Publications and source records attributed to L T Potter.

At least 55 records · Page 3Linked to original sources
PubMed1981

A minor component of the binding of [3H]guanyl-5'-yl imidodiphosphate to cardiac membranes associated with the activation of adenylate cyclase.

Incubation of a preparation of cardiac plasma membranes with 20 microM (--)-isoproterenol and 1 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) results in prolonged and maximal activation of adenylate cyclase and prolonged binding of [3H]Gpp(NH)p to the membranes. The total number of [3H]Gpp(NH)p sites must exceed those required for enzyme activation (N-sites) since the rate of binding is much slower than the rate of activation. Attempts to measure the amount of [3H]Gpp(NH)p released from N-sites during incubation of membranes with isoproterenol and GTP were frustrated by limited (about 20%) reversal of binding and of enzyme activation. However, the amount of [3H]-Gpp(NH)p bound to membranes could be reduced by preincubating them in a concentration of App(NH)p (0.1-0.5 mM) which does not interact with the N-site and most of the [3H]Gpp(NH)p subsequently bound could be removed by further incubation of the membranes with EDTA, propranolol, and Gpp(NH)p. Under these conditions, full enzyme activation was retained and was associated with residual bound [3H]Gpp(NH)p of about 0.3 pmol/mg of protein. This amount equals the concentration of beta-adrenoreceptors; and the rate and Kd (0.22 microM) for the binding residual [3H]-Gpp(NH)p approximate the rate and Ka for enzyme activation. The rate of enzyme activation and [3H]-Gpp(NH)p binding to the residual component was increased in the presence of isoproterenol. Competition binding studies for the residual component revealed that Gpp(NH)p congruent to GTP greater than GDP greater than GMP. The results suggest that residual [3H]Gpp(NH)p binding is to the adenylate cyclase-coupled guanine nucleotide binding protein.

Adenylyl Cyclases↗
PubMed1980

Effect of propranolol on beta-adrenoceptors in rat hearts.

The question of whether the chronic administration of propranolol modifies the numbers or properties of cardiac beta-adrenoceptors was examined because of many reports suggestive of cardiac hypersensitivity following the withdrawal of beta-antagonists. In three studies, rats were given (+/-)-propranolol, 30 mg/day, orally or intraperitoneally, for 1 to 7 weeks. The numbers and affinities of specific binding sites for radioactive dihydroalprenolol in whole hearts from 60 control and 75 test animals were found to be the same. Thus catecholamine deprivation does not exert an important regulatory effect on most beta-receptors in the heart, as it does in the brain.

Alprenolol↗
PubMed1980

Distribution and function of beta-adrenoceptors in different chambers of the canine heart.

1 An improved binding assay involving (-)-[(3)H]-dihydroalprenolol (DHA) and KCl-washed cardiac membranes was developed to study beta-adrenoceptors in the canine heart quantitatively.2 Receptor numbers varied from 3.8 to 7.1 pmol/g fresh tissue, showing a steady increase from left atrium --> right atrium --> right ventricle --> interventricular septum --> left ventricle. With one minor exception, the same pattern was found for adenylate cyclase activity and Na(+), K(+)-activated ATPase activity.3 The binding of DHA was inhibited in the expected manner by beta-adrenoceptor agonists and antagonists, and was stereospecific, in confirmation of previous studies. Dissociation constants determined from Scatchard analyses included DHA: 2.5 nM; (-)adrenaline: 230 nM; (-)noradrenaline: 1167 nM. Kinetic analyses of the binding of DHA yielded a K(D) of about 4 nM.4 The distribution of beta-receptors is closely related to that of blood flow and the arrival plus retention of a circulating catecholamine, but is markedly different from that of endogenous noradrenaline, and thus adrenergic nerve terminals. Most receptors thus appear not at synapses but diffusely localized where they can react with circulating adrenaline.5 Evidence is discussed that beta-receptors at synapses respond primarily to neural noradrenaline, less to circulating adrenaline, and hardly at all to circulating noradrenaline; responses mediate increased cardiac output during exercise. In contrast most cardiac beta-receptors appear to respond only to adrenaline, and to be used, except at times of severe circulatory stress, during psychological stress.

Adenylyl Cyclases↗
PubMed1980

Cardiac beta-adrenoceptors during normal growth of male and female rats.

1 A binding assay involving (-)-[3H]dihydroalprenolol (DHA) and KCl-washed cardiac membranes were used to assess the numbers and affinities of beta-adrenoceptors in hearts from male and female rats varying in age from about 2 weeks to 18 months. 2 Although female rats grow more slowly and attain lower adult weights than male rats, heart weights increased in approximate proportion to body weight with little sex difference. 3 As heart weight increased about three fold, beta-receptors increased three fold. Since the number of myocardial cells is believed to be nearly constant during postnatal growth, the numbers of receptors/cell presumably increases similarly. 4 As heart weight increased, the number of beta-receptors per g of tissue decreased according to the equation: total pmol/g = 4.33 - 1.43 x heart weight, equally in males and females. 5 Dissociation constants for DHA (2 to 4 nM) remained the same, and equal, in male and female rats during their growth. 6 An excellent correlation was found between the decline in beta-receptors/g tissue during growth and the decline in the area of the external sarcolemma/g tissue. The data suggest that the number of receptors per unit area remains constant during growth, and thus that cell surface area is a major factor determining normal numbers of receptors per cardiocyte.

Animals↗
PubMed1980

Purification and partial characterization of cardiac plasma membranes rich in beta-adrenoreceptors.

A procedure was developed to purify plasma membranes from cardiac tissue. Canine ventricular tissue was homogenized relatively gently in hypotonic buffer, extracted briefly with 0.15 M sodium perchlorate at 4 degrees C, and membranes were collected in an intermediate pellet by centrifugation at 27,500 X gmax. At this stage, most contractile proteins were removed (although Z-band structures were apparent), and the recoveries of Na+, K+-ATPase, beta-adrenoreceptors and 5'-nucleotidase were 40--50%. Treatment of these particles with 0.5% dextran sulfate and continuous density gradient centrifugation resulted in the separation of membranes having 111 and 3.6 pmol/mg protein specific binding sites for [3H] ouabain and [3H] dihydroalprenolol, respectively. Electron micrographs showed medium-sized vesicles. 5'-Nucleotidase was also markedly purified but could be partially separated from the other plasma membrane markers in continuous sucrose gradients. Enzymes associated with mitochondria, lysosomes, and endoplasmic reticulum were efficiently removed. SDS-polyacrylamide gels showed six major and many minor bands. Adenylate cyclase remained responsive to hormones and Gpp(NH)p, but little purification was apparent. The membrane preparation has proved useful for studies of a nucleotide-binding protein associated with cyclase activation.

Adenylyl Cyclases↗
PubMed1978

beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle.

Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.

Adenylyl Cyclases↗
PubMed1977

Postsynaptic membranes in the electric tissue of Narcine: I. Organization and innervation of electric cells. Fine structure of nicotinic receptor-channel molecules revealed by transmission microscopy.

The ventral, postsynaptic membranes of the electroplaques of Narcine were examined for structures corresponding in location, numbers, position and form with nicotinic acetylcholine receptor-channel molecules. Electron micrographs of conventionally fixed material showed that the thickened outer lamina of these membranes contained about 6100 stained structures/micron 2; and calculations from this packing density and the estimated area of postsynaptic membranes/g tissue yielded a total number of structures within a factor of two of the number of receptor molecules/g determined from the binding of alpha-bungarotoxin. Examination of tissue prepared with a tannic acid/glutaraldehyde fixative revealed that the structures were uniform in character, cylindrical in shape, transmembrane in position, and that each contained a central core. Their diameter matched that of isolated receptor molecules, and their size can account for most of the receptor protein. We conclude that these receptor molecules are largely intramembranous, cylindrical and exhibit transmembrane pores.

Animals↗
PubMed1977

Postsynaptic membranes in the electric tissue of Narcine: II. A freeze-fracture study of nicotinic receptor molecules.

The ventral, postsynaptic membranes of the electroplaques of Narcine were found to containe intramembranous particles similar in location, packing density (about 5700/micron 2), transmembrane position and end appearance to nicotinic acetylcholine receptor-channel molecules. In fixed tissue the particles were limited to the cytoplasmic lamina, while in unfixed tissue an equivalent number were found symmetrically in both laminae. Four populations of particle diameters were observed in each unfixed lamina, even though other morphological evidence indicates the presence of large number of molecules of uniform structure, and biochemical studies of isolated postsynaptic membranes indicate that at least 70% of the membrane protein is receptor-channel protein. Intramembranous particles in dorsal, non-innervated electroplaque membranes, presumably representing Na+, K+-associated ATPase and other channel proteins, were found to have similar characteristics to particles in ventral membranes. Receptor-channel molecules cannot, therefore, be distinguished from other intrinsic membrane proteins by freeze-replication alone.

Animals↗
PubMed1977

Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization.

Postsnyaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9--10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50% of the tissue toxin-sites, 5% of the protein, 4% of the ATPase and less than 2% of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by 'differential' centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.

Animals↗
PubMed1977

Postsynaptic membranes in the electric tissue of Narcine: IV. Isolation and characterization of the nicotinic receptor protein.

The nicotinic receptor protein of the electric tissue of Narcine was purified in several different media by partial isolation of postsynaptic membranes and affinity chromatography. Protease inhibitors were found to be necessary to prevent degradation of the protein, and both EDTA and Tris buffer were used in addition to prevent intramolecular crosslinking of 44,000 and 58,000 dalton subunits by tissue factors. The intact protein was found to have a molecular weight close to 400,000, and appears to be composed of four subunits of 44,000 daltons, two to three of 48,000, one of 58,000 and one of 65,000. All the subunits are glycoproteins and their amino acid compositions show similar hydrophobicity and acidity, suggesting similar positioning in postsynaptic membranes. Crosslinking experiments showed that acetylcholine and alpha-bungarotoxin bind to the smallest subunit, and suggest the juxtaposition of at least two of these subunits, and of all four toxin molecules bound to a receptor molecule. Morphological studies of the protein in membranes and after purification indicated cylindrial molecules with central cores.

Acetylcholine↗