Calcium-independent stimulation of glycogenolysis by arginine vasotocin and catecholamines in liver of the axolotl (Ambystoma mexicanum) in vitro.

Arginine vasotocin (AVT) caused a concentration-dependent increase of glycogen phosphorylase alpha activity, breakdown of glycogen and release of glucose, when added to pieces of axolotl liver in organ culture. The concentration causing half-maximal response (EC50) was about 1 nmol/l. These actions of AVT were unaffected by the adrenergic antagonists propranolol, yohimbine and prazosin, but were blocked by equimolar amounts of d(CH2)5Tyr(Me)AVT, a synthetic antagonist of vasopressin. Arginine vasotocin similarly caused glycogenolysis in isolated perfused axolotl liver where the EC50 was about 0.1 nmol/l. The glycogenolytic action of AVT (10 nmol/l) was sustained for at least 3 h in Ca2+-free perfusion and longer in organ culture. No increase in Ca2+ concentration in the effluent perfusion medium was apparent during AVT-induced glucose release. Omission of Ca2+ from the medium, together with addition of EGTA (2.5 mmol/l) to the organ culture, had only a slight inhibitory effect upon the rate of glycogenolysis brought about by AVT and did not inhibit the glycogenolytic action of catecholamines. Addition of the calcium ionophore A23187 (5 mumol/l) neither caused glucose release nor abolished the glycogenolytic action of AVT added subsequently. Nevertheless, A23187 caused increased loss of 45Ca from Ca2+-loaded liver pieces whereas AVT was without effect. There was a slight accumulation of cyclic AMP (cAMP), but not cGMP, in axolotl liver pieces cultured in the presence of 0.1 mumol AVT/l and this was accentuated in the presence of phosphodiesterase inhibitors. We conclude that, in contrast to the position in mammals, Ca2+ is not involved in the glycogenolytic actions of AVT or catecholamines in axolotl liver. Preliminary experiments suggest that the same is true in the carp and we suggest that the involvement of Ca2+ in regulation of hepatic glucose release may not have evolved until after the amphibians separated from the ancestors of the mammals.

Coupled diminished energy turnover and phosphorylase a formation in contracting hypothyroid rat muscle.

Hypothyroidism leads to a diminished phosphorylase a formation and a reduction in both glycogen breakdown and lactate production during tetanic stimulation of fast-twitch skeletal muscle (mixed type). Phosphorylase kinase activity is almost 50% lower in the hypothyroid (Tx) group, and the possibility that this might explain the reduction in phosphorylase a formation is discussed. In both the Tx group and the euthyroid (C) group the reconversion of phosphorylase a to b correlates well with a decrease in the energy cost for contraction.

The phosphorylase kinase activity of hearts from phosphorylase kinase-deficient mice.

In an assay measuring radioactive incorporation from gamma--P32P]ATP into phosphorylase b, cardiac muscle extracts from mice with the phosphorylase kinase deficiency mutation showed significant, calcium-dependent phosphorylase kinase activity that was 10 to 15% of that of Swiss mice, the control strain. Isoproterenol stimulated significant phosphorylase a accumulation in both isolated atria and right ventricular strips of phosphorylase kinase-deficient mice, and the drug-stimulated increases in phosphorylase a activity the the contractile responses of right ventricular strips were similar in Swiss and phosphorylase kinase/deficient mice.

Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts.

In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6-phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity.

The sarcoplasmic reticulum-glycogenolytic complex in mammalian fast twitch skeletal muscle. Proposed in vitro counterpart of the contraction-activated glycogenolytic pool.

Evidence is presented that the sarcoplasmic reticulum (SR)-glycogenolytic complex isolated from fast twitch skeletal muscle is a highly specific, functionally defined compartment for phosphorylase regulation. The addition of ATP alone results in prompt phosphorylase activation which demonstrates calcium dependence similar to the calcium-magnesium ATPase that catalyzes SR calcium transport suggesting that these two calcium-requiring -ystems might interact within the complex. Lowering extravesicular calcium concentration by transport of calcium into the SR lumen resulted in inactivation of phosphorylase a. This effect could be prevented by the addition of the calcium ionophore X537A which inhibits SR calcium sequestration or a calcium EGTA buffer which maintains free calcium. It was mimicked by EGTA addition. Since exogenous phosphorylase b and phosphorylase a were not activated or inactivated, respectively, by the endogenous activating enzymes or phosphatase in the SR-glycogenolytic complex, these regulatory enzymes may be compartmented. In addition, endogenous phosphorylase could be uncoupled from its activating enzymes by amylase treatment. These results suggest that the SR-glycogenolytic complex in fast twitch skeletal muscle is a compartmented system for phosphorylase activation controlled by SR calcium flux, a feature in contrast to the cardiac complex (Entman, M.L., Kaniike, K., Goldstein, M.A., Nelson, T.E., Bornet, E.P., Futch, T.W., and Schwartz, A. (1976) J. Biol. Chem. 251, 3140-3146). We suggest that the complex is the in vitro counterpart of the well documented rapid burst of glycogenolysis which ensures with the onset of contraction.

Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KCl and carbachol.

It has been proposed that Ca2+-dependent myosin light chain (P-light chain) phosphorylation in smooth muscle permits cycling of myosin cross-bridges within myofibrillar elements for muscle shortening, but a second Ca2+-dependent regulatory mechanism is responsible for force generation. Accordingly, we examined P-light chain phosphorylation and another Ca2+-dependent protein phosphorylation reaction, phosphorylase a formation, in bovine tracheal smooth muscle during isometric force generation elicited by the cholinergic agonist carbachol or KCl depolarization, two stimuli thought to increase the concentration of sarcoplasmic free Ca2+ by mobilizing different pools of Ca2+. Increases in P-light chain phosphorylation reached maximal values of 0.79 and 0.59 mole of phosphate per mole of P-light chain at 1 min and then declined during maintained isometric force developed in response to 1 microM carbachol and 60 mM KCl, respectively. Carbachol elicited approximately twice the amount of force as found in the presence of KCl, and yet a more rapid rate of decline in the phosphate content of P-light chain was apparent. Decreases in maximal levels of phosphorylase a also occurred during carbachol-mediated isometric force maintenance, yet did not occur with KCl stimulation. Concentration-dependent responses with carbachol and KCl showed a positive relationship between the extent of P-light chain phosphorylation and extent of developed isometric force after 1 min of contraction with both stimuli. Under no conditions was force generated without P-light chain phosphorylation. The concentration dependence of phosphorylase a formation with KCl was similar to isometric force and P-light chain phosphorylation. However, concentrations of carbachol necessary to stimulate phosphorylase a formation were much higher than those required for stimulation of isometric force and P-light chain phosphorylation. Furthermore, carbachol attenuated the stimulation of phosphorylase a formation by isoproterenol. Thus, carbachol appears to have both an inhibitory and stimulatory effect on phosphorylase a formation in bovine tracheal smooth muscle. These results also indicate that maintained isometric force in smooth muscle may be dependent upon the maximal extent of P-light chain phosphorylation obtained during an early temporal transient in phosphorylation.

Rate dependence of isoproterenol-stimulated phosphorylase a formation in mouse heart.

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, isoproterenol (3 microM) stimulation of phosphorylase a accumulation was approximately a linear function of the rate of contraction from 0.2 to 5 Hz. In muscles incubated in the absence of isoproterenol, the phosphorylase a activity did not increase when the contraction frequency was varied over this range. At a low frequency of stimulation (0.2 Hz), phosphorylase a activity was not increased after a 5-min exposure to 3 microM isoproterenol, as compared to a 4-fold increase in phosphorylase a activity at a high frequency (3.-3 Hz). Isoproterenol (3 microM) increased tissue cyclic AMP content and the activated form of phosphorylase kinase activity to similar extents at both frequencies. N6,O2'-dibutyryl cyclic AMP increased the phosphorylase a activity at both frequencies but the increase at 3.3 Hz was approximately 3-fold greater than at 0.2 Hz. Verapamil did not block isoproterenol-stimulated phosphorylase a activity at 3.3 Hz at a concentration (0.6 microM) that inhibited the increased sensitivity to the inotropic action of isoproterenol seen at high frequencies of contraction. Isoproterenol stimulation of phosphorylase a accumulation did not correlate with developed tension. It is proposed that the difference in isoproterenol stimulation of phosphorylation b to a conversion at 0.2 and 3.3 Hz primarily results from a difference in Ca++ control of the activity of the activated form of phosphorylase kinase.

Ultraviolet radiation-induced decreases in tension and phosphorylase a formation in rat aorta.

Helical strips of rat aorta contracted with norepinephrine (10 nM) respond to ultraviolet (UV) radiation (340-360 nm) with a transient decrease in tension. The UV radiation-dependent relaxation is completely reversible and endothelial cell-independent. Although decreased tension is associated with a rise in tissue cGMP content, the cAMP level is unchanged after UV radiation. A significant inhibition of phosphorylase a formation which occurs coincidently with the rise in cGMP and decline in tension is observed with UV radiation. The effects of UV radiation on cGMP, phosphorylase a formation and tension were blocked by methylene blue. Relaxation and inhibition of phosphorylase a formation in response to UV radiation were also partially reversed with higher concentrations of norepinephrine (100 nM). Our results suggest that cGMP may mediate UV radiation-dependent reactions by reducing cytoplasmic Ca2+.

Characteristics of acetylcholine-induced phosphorylase a activity in uterine segments as a substitute for contractile response to acetylcholine.

Studies were made on whether the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh. This ACh-induced phosphorylase a activity was dependent upon the concentration of ACh and was inhibited by atropine, suggesting that it was linked to muscarinic ACh receptors. Both extracellular calcium and an increase of the intracellular calcium concentration were needed for its activation by ACh. Ca2+-antagonists such as Co2+, diltiazem, nitrendipine and verapamil inhibited the ACh-induced activity, suggesting that the activation by ACh required the influx of calcium ions into the uterine muscle through Ca2+-antagonist sensitive Ca2+ channels. The IC50 values of CoCl2, diltiazem, nitrendipine and verapamil on the ACh-induced phosphorylase a activity were 3.4 x 10(-3) M, 2.5 x 10(-4) M, 2.5 x 10(-5) M and 1.1 x 10(-4) M, respectively. These values were comparable with the IC50 values of these Ca2+-antagonists on the contractile response of isolated rat uterine muscle segments to 3 x 10(-4) M ACh. The inhibitory effects of Co2+, nitrendipine and verapamil, but not diltiazem, on ACh-induced phosphorylase a activity were attenuated by higher concentrations of CaCl2 (0.36 to 2 mM). These findings suggested that the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh.

Effects of nitroprusside, glyceryl trinitrate, and 8-bromo cyclic GMP on phosphorylase a formation and myosin light chain phosphorylation in rat aorta.

The effects of nitroprusside (NP), glyceryl trinitrate (GTN), and the 8-bromo analog of cyclic GMP (8-Br-cGMP) on norepinephrine (NE)-stimulated phosphorylase a formation and myosin light chain (MLC) phosphorylation were examined in the rat aorta. NE produced a time-dependent increase in tension, phosphorylase a formation, and MLC phosphorylation. The formation of phosphorylase a and phosphorylation of MLC were transient, since both processes declined to basal levels within 30 min after the addition of NE even though tension remained elevated. NP and GTN inhibited tension, phosphorylase a formation, and MLC phosphorylation although inhibition of phosphorylase was greater when strips were treated with submaximal (i.e., 0.01 microM) NE concentrations. GTN was a more effective inhibitor of phosphorylase a formation than NP in NE-treated strips, although both agents and 8-Br-cGMP inhibited MLC phosphorylation. The guanylate cyclase inhibitor methylene blue (10 microM) effectively prevented the effects of NP and GTN. The results suggest that NP, GTN, and 8-Br-cGMP inhibit phosphorylase kinase and MLC kinase activation by lowering Ca2+ in the cell. This hypothesis is supported by the observations that 8-Br-cGMP inhibited the Ca2+-dependent, KCl-induced phosphorylase a formation most markedly at reduced concentrations of extra-cellular Ca2+. In addition, neither NP, GTN, nor 8-Br-cGMP inhibited phosphorylase a formation in forskolin-treated tissues, which occurred in response to cAMP-dependent phosphorylation of phosphorylase b kinase.

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle.

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.

Regulation of phosphorylase A formation and calcium content in aortic smooth muscle and smooth muscle cells: effects of atrial natriuretic peptide II.

Atrial natriuretic peptide II (ANP II) raises cyclic GMP and relaxes vascular smooth muscle in vitro. The manner in which ANP II relaxes vascular smooth muscle is unknown but may involve alterations in the concentration of free intracellular Ca++. To examine this possibility, changes in intracellular Ca++ were monitored in rat aortic strips using the Ca++-dependent conversion of phosphorylase b to a, while Ca++ levels and phosphorylase were measured in cultured rat aortic smooth muscle cells. ANP II produced time- and concentration-dependent decreases in phosphorylase a and tension in norepinephrine-contracted aortic strips. The decrease in the formation of phosphorylase a was accompanied by an increase in cyclic GMP content. ANP II also decreased phosphorylase a formation in K+-depolarized tissues but to a lesser extent. Agonists such as angiotensin II and arginine vasopressin, and depolarizing concentrations of K+ elevated Ca++ levels in cultured aortic cells. ANP II inhibited Ca++ accumulation to either agonists or K+, but was more effective against agonists. Phosphorylase a formation which was increased by agonists and K+ in cultured cells was also inhibited by ANP II. We conclude that phosphorylase a formation can be a useful indicator of intracellular Ca++ concentrations in smooth muscle preparations and that ANP II regulates Ca++ levels in agonist and depolarized smooth muscle, suggesting that ANP II affects mainly Ca++ removal from the cytoplasm.