H De Wulf

(3H)-vasopressin binding to isolated rat hepatocytes and liver membranes: regulation by GTP and relation to glycogen phosphorylase activation.

Specific vasopressin binding to rat hepatocytes and rat liver membranes was measured using biologically active (3H)-Tyr2-Lys8-vasopressin (8.5 Ci/mM). In both systems, vasopressin binding was found to be time-dependent, reversible, and saturable. The kinetic parameters for vasopressin binding were: apparent dissociation constants (Kd): 4.9 nM and 15 nM; maximal binding capacities: 0.83 pmoles/mg protein and 2.10(5) sites/Cell for purified membranes and intact cells respectively. The relative affinities of 19 vasopressin structural analogues were deduced from competition experiments and compared to the previously determined glycogenolytic (or antiglycogenolytic) potencies of these analogues. For both agonists and antagonists, a highly significant correlation was demonstrated between pKd and pKa (or pKi) values, suggesting that the detected binding sites are the physiological receptors involved in the glycogenolytic action of vasopressin on the rat liver. The affinity of antagonists for binding to these receptors is the same for both membranes and cells. In contrast, agonists which bind to vasopressin receptor sites have a higher affinity for purified membranes than for intact cells (Kd membranes/Kd cells = 8 +/- 1). GTP (0.1mM) reduced the affinity of agonists but not of antagonists for binding to membranes and abolished the differences between Kd values for binding to hepatocytes and membranes.

The activation of liver glycogen phosphorylase by vasopressin

It has been shown [ 1 ] that vasopressin stimulates glycogenolysis in the rat liver at concentrations which can occur in vivo, especially during haemorrhagic shock. We report here that the glycogenolytic effect of vasopressin is due to the activation of glycogen phosphorylase, the rate limiting enzyme in glycogen degradation. The mechanism involved appears different from that of glucagon or adrenaline: it is apparently not mediated by cyclic AMP, since in contrast with the two latter hormones no increase in protein kinase activity is observed with vasopressin. A preliminary communication describes part of these results PI.

Extracellular ATP and UTP exert similar effects on rat isolated hepatocytes

1 Extracellular UTP and ATP show obvious similarities in their control of several metabolic functions of rat isolated hepatocytes. 2 They have a similar time‐course and concentration‐dependency for the activation of glycogen phosphorylase, the generation of inositol trisphosphate (IP3), the inhibition of glycogen synthase and the lowering of adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) levels. 3 There is a similar synergism of the nucleotides with glucagon in activating phosphorylase. 4 They undergo a similar inhibition by phorbol myristic acid of their glycogenolytic effect. 5 The ATP and UTP effect on IP3 levels are not additive. 6 It is tentatively concluded that UTP and ATP use a common receptor.

The activation of liver phosphorylase b kinase by glucagon

Although cyclic AMP was discovered in studies of the mode of action of glucagon in promoting liver glycogenolysis (see [l] ), the entire sequence of events initiated by the cyclic nucleotide is less well known in liver than in muscle. Indeed, in this tissue, as has been worked out very well by Krebs and co-workers (see [2] ) cyclic AMP brings about a sequential activation of protein kinase, phosphorylase kinase and glycogen phosphorylase, leading ultimately to an enhanced rate of glycogen breakdown. In liver, the activation of glycogen phosphorylase by glucagon also involves a rise in the cyclic AMP content (see [ 11) and an activation of protein kinase [3,4] but up till now, there has only been one report describing an enhanced activity of phosphorylase kinase [5]. In the present report, we show that glucagon does indeed cause a substantial increase in the liver phosphorylase kinase activity; we show furthermore that this activation is caused by the action of the cyclic AMP dependent protein kinase.

The activation of liver glycogen phosphorylase by angiotensin II.

Vasopressin, in amounts likely to be produced in haemorrhagic shock, causes glycogenolysis in the liver [ 1,2] through a prompt activation of liver glycogen phosphorylase (EC 2.4.1 .l) [3,4] . This activation, unlike that produced by glucagon or adrenaline, is not mediated by an enhanced activity of protein kinase (EC 2.7.1.37) [3] and neither does vasopressin increase the concentration of cyclic AMP in rat liver [5] . A haemorrhage results in an increase of the blood levels of angiotensin II (for recent reviews see [6,7 ] ) and the question arises naturally whether angiotensin II shares the glycogenolytic properties of vasopressin. Conflicting reports can be found in the literature concerning an effect of angiotensin II on blood sugar level, which has been reported to rise [g-10] , to remain unaltered [l I-131 or even to decline [14] after the injection of the octapeptide. It should however been noted that those authors who did not find a hyperglycemic response to angiotensin II [ 1 1 141 have been using fasting animals or subjects, so that the lack of a systemic hyperglycemia can conceivably stem from unsufficient liver glycogen stores. It has indeed been shown [8] that the hyperglycemia caused by angiotensin II is accompanied with a progressive loss of liver, but not of muscle, glycogen. Furthermore, two groups of investigators have presented evidence for a direct glycogenolytic effect of angiotensin II on isolated rat liver preparations [8,10]. We report here that, as vasopressin, angiotensin II causes a direct activation of liver glycogen phosphorylase, the rate limiting enzyme for glycogenolysis; likewise, the mechanism involved

Characterization of the effects of adenosine 5′‐[β‐thio]‐diphosphate in rat liver

1 In rat liver cells micromolar concentrations of adenosine 5′‐[β‐thio]diphosphate (ADPβS), activate glycogen phosphorylase by an adenosine 3′:5′‐cyclic monophosphate (cyclic AMP)‐ independent mechanism. 2 As with adenosine 5′‐triphosphate (ATP), ADPβS also inhibits the rise in cyclic AMP after glucagon. 3 Cytosolic Ca2+ measured in single cells is rapidly increased with a pattern similar for ADPβS and for ATP. 4 At variance with ATP, ADPβS hardly increases inositol 1,4,5‐trisphosphate (IP3) levels. 5 Phorbol myristic acetate, which inhibits only slightly the glycogenolytic effect of ATP, almost completely abolishes this effect of ADPβS. 6 With adenosine 5′‐[β‐[35S]thio]diphosphate (ADPβ[35S]) as radioligand, we detected specific purinoceptors on rat liver plasma membranes. Binding consists of a major binding component with KD = 0.7 μm and Bmax = 51 pmol mg−1 of protein, probably mediating the activation of glycogen phosphorylase, and a minor high affinity, low capacity binding component with no obvious function. 7 It is concluded that the differences in biological effects between ATP and ADPβS may involve different receptors and/or different transduction mechanisms and that ADPβ[35S] can be used to detect the specific binding sites for ADPβS.

REGULATION OF BRAIN GLYCOGENOLYSIS: cAMP DEPENDENT AND INDEPENDENT MECHANISMS

Publisher Summary This chapter discusses a study to examine effect of biogenic amines and depolarization on the production of cAMP and on the activity of phosphorylase kinase and glycogen phosphorylase in mouse (NMRI) brain. The addition of norepinephrine (NE), 5-hydroxytryptamine, and adenosine induced a 2-fold activation of glycogen phosphorylase within 30 s. NE elicited a 3- to 4-fold increase in the level of cAMP, of which only a fraction was necessary to achieve the observed phosphorylase activation. Tetrodotoxin prevented the accumulation of cAMP and the activation of glycogen phosphorylase by veratridine but not the enzymic activation by K + depolarization. Glycogenolysis in the brain is regulated by neurotransmitters and depolarization through a cAMP dependent and through a cAMP independent way, probably with Ca ++ as the intracellular mediator is also proposed.