On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

Localization of the catalytic subunit of a cyclic AMP-dependent protein kinase(S) and acceptor proteins on the external surface of the fat cell membrane.

Cyclic AMP in μM concentrations increases the labeling of a membrane component of approximately 53,000 daltons as well as the labeling of a minor peptide of 18,000 daltons when isolated, intact rat fat cells are incubated with μM concentrations of (γ-³²P)ATP. Controlled tryptic digestion of intact cells followed by incubation with (γ-³²P)ATP results in diminution of labeling of both of these phosphopeptides indicating susceptibility, hence, access of either the catalytic site or the substrates to trypsin action. Addition of the catalytic subunit of the cyclic AMP-dependent protein kinase from beef heart to cells previously treated with trypsin results in the labeling of both phosphopeptides comparable to untreated cells. These findings indicate the catalytic subunit(s) of the cyclic AMP-dependent protein kinase(s) as well as these two phosphopeptides of the intact rat fat cell must be located on the external surface of the plasma membrane. Further, the catalytic subunit(s) of the membrane-located cyclic AMP-dependent protein kinase(s) is susceptible to trypsin action whereas the membrane peptides serving as substrates are not.     

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Purification and succinylation of cyclic GMP from large volume samples and radioimmunoassay of succinyl cyclic GMP.

Improved procedures for isolation of cyclic GMP and cyclic AMP and radioimmunoassay of cyclic GMP with succinylation are described. Procedures involved include modified chromatography on alumina and succinylation of cyclic GMP followed by purification of succinyl cyclic GMP on a Dowex AG 1×8 column. These procedures are convenient and applicable to any volume up to 50 ml of tissue extracts and especially for isotonic incubation mixtures. This assay system is sensitive to 6 femtomoles of cyclic GMP/tube. On radioimmunoassay, free and antibody bound [¹²⁵I]-labeled cyclic GMP are separated by Millipore filtration. Cyclic GMP levels in several tissue samples were determined in order to show the applicability of the procedures.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Enzymatic activities of coated vesicle fractions from bovine and rat cerebrums

The changes in the enzymatic properties of coated vesicle fractions obtained from bovine cerebrums and rat forebrains were investigated as the preparation procedures were modified. Because Mg²⁺-dependent ATPase activity in the coated vesicle fractions that were prepared by conventional centrifugation methods appeared to derive from contaminating particulates, the activity was examined after further purification by column chromatography and confirmed by histochemical technique. Both the coat proteins and the vesicles enclosed in the coat networks failed to show the activity. Since plain synaptic vesicles are known to have ATPase activities, the results may indicate that the membrane structure of synaptic vesicles is modified between the coated vesicle stage and the plain vesicle stage of vesicle recycling as Heuser and Reese proposed (J. Cell Biol.57, 315–344, 1973). The comparison of the specific activity change in ATPase with those of acetylcholinesterase and NADPH-cytochrome c reductase suggested that there were two types of microsomes contaminating coated vesicle fractions that were prepared conventionally.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles

The Mg²⁺-dependent, K⁺-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H⁺ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth).Valinomycin stimulated both dye uptake and K⁺-ATPase (valinomycin-stimulated K⁺-ATPase); stimulation by valinomycin was due to increased K⁺ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K⁺-ATPase and H⁺ accumulation as an index, the sequence for anion permeation was NO₃^? > Br^? > Cl^? > I^? > acetate ≈ isethionate. When permeability to both K⁺ and H⁺ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K⁺-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an ‘uncoupling’ of ATP hydrolysis from H⁺ accumulation.Thiocyanate interacts with valinomycin inhibiting the typical action of the K⁺ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN^? to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K⁺-ATPase, it appears that SCN^? is a very     

Myocardial muscarinic acetylcholine receptor: Choline and tris unmask heterogeneity of antagonist binding sites

The binding properties of myocardial muscarinic acetylcholine receptors are altered in the presence of choline or Tris. The binding of the antagonist [³H]quinuclidinyl benzilate is reduced in the presence of choline or Tris buffer, when compared to parallel determinations in a physiologic salt solution or phosphate buffer. Scatchard analysis indicates the reduced binding is due to a decrease in the apparent number of receptor sites. Experiments with other organic buffers exclude the possibility that the reduced binding in Tris is due to the absence of sodium ions. In the presence of choline or Tris up to 45% of the receptors are not accessible to [³H]quinuclidinyl benzilate. The remaining sites maintain their high affinity for the antagonist. A heterogeneity of antagonist sites is evident.