Agostino Trevisani

The role of lipids in the activity of adenylate cyclase of rat liver plasma membranes

Abstract The effect of mild lipid extraction and phospholipases on isolated rat liver plasma membrane was investigated. Extraction of membrane at 4 ° with a shaked mixture of petrol ether- n -butanol resulted in a dramatic reduction of the concentration of phosphatidylethanolamine (−97%) and phosphatidylserine (−99%), whereas phosphatidylinositol was reduced to about 30% of the initial value. Simultaneously the activity of adenylate cyclase as well as that of Mg 2+ -ATPase was strongly reduced −83 and −85%, respectively); another membrane enzyme, 5′-nucleotidase was slightly stimulated. The activity of solvent-treated adenylate cyclase was partially restored by addition of total membrane phospholipids or phosphatidylinositol, while phosphatidylserine was ineffective. Both phospholipase A and C reduced adenylate cyclase activity. However the effect of phospholipase C was prevented by serum albumin, while that of phospholipase A was not. This suggests that the effect of the latter enzyme may be related to alterations of phospholipids and (or) of membrane structure.

Role of serotonin and cyclic AMP on facilitation of the fast conducting system activity in the leechHirudo Medicinalis

In the nervous system of the leech Hirudo medicinalis it has been possible to study short-term plastic changes. Depression and facilitation have been demonstrated in the fast conducting system (FCS) activity; this pathway consists of a chain of electrically linked neurons present in each ganglion. In semi-intact animals or in preparation of nerve cord and segments of body wall, both electrical stimulation of peripheral roots and tactile stimulation of the skin induced, after repetitive stimulation (0.1/s) a prolonged decrement of FCS response. Strong nociceptive stimulation applied onto the head or the body wall produced a sustained facilitation of the waned response. The same potentiation has been observed by perfusing the isolated ganglion with serotonin (5 x 10(-5) M). Such a potentiation is abolished by preincubation with methysergide, an antagonist of serotonin, and with imidazole, a cAMP-phosphodiesterase activator. Such an effect is mimicked by an analog of cAMP, db-cAMP. Simultaneous recordings of both T neurons (intracellularly) and FCS firing discharge showed that, during FCS response decrement, the T cell activity remained unchanged and no modification of conductance occurred, excluding therefore a detectable involvement of sensory neurons in the depression. These results suggest that short-term plastic changes of the FCS of the leech are due to a prolonged potentiation of synaptic transmission as a result of serotonin-mediated increase in cAMP.

The role of phosphatidylserine in the hormonal control of adenylate cyclase of rat liver plasma membranes

The involvement of phospholipids in the responsiveness of adenylate cyclase of rat liver plasma membranes to hormones was studied, using a mild extraction procedure and phospholipase A and C treatment for the removal of membrane phospholipids. Mild extraction of membranes caused a reduction in the basal activity of adenylate cyclase which was also no longer sensitive to hormones and F−. Addition of phosphatidylserine almost completely restored the epinephrine responsiveness of the enzyme, but only partially restored the glucagon and F− sensitivity. Phosphatidylinositol, which restored the basal cyclase activity, had no effect on hormonal and F− sensitivity. Phosphatidylethanolamine and phosphatidylcholine had no effect. Treatment of membranes with phospholipase A and C abolished the hormonal and F− responsiveness of adenylate cyclase; phospholipase A also decreased its basal activity. The hormonal and F− responsiveness were partially reactivated with phosphatidylserine, but not with phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. These observations are consistent with the catalytic and regulatory subunits of plasma membrane adenylate cyclase both being lipoproteins, the former requiring phosphatidylinositol and the latter phosphatidylserine for proper functioning.

Modulation by cyclic AMP in vitro of liver plasma membrane (Na+—K+)-ATPase and protein kinases

Abstract The effect of cyclic AMP on (Na+—K+)-ATPase and protein kinase activities of isolated liver plasma membrane has been investigated. Theophylline and caffeine, several cyclic nucleotides (including cyclic AMP, dibutyryl cyclic AMP, cyclic GMP and cyclic UMP), and sodium butyrate reduced the activity of (Na+—K+)-ATPase. The most powerful inhibitor was dibutyryl cyclic AMP; however, its action is probably due to the combined effects of cyclic AMP and butyrate. The blocking agent propranolol prevented the action of both epinephrine and cyclic AMP. Liver plasma membranes contain protein kinase(s), which catalyze the transfer of terminal phosphate of ATP into protein of plasma membrane itself (endogenous phosphorylation) as well as into added proteins (exogenous phosphorylation). Endogenous phosphorylation was inhibited by cyclic AMP, dibutyryl cyclic AMP and cyclic GMP. On the other hand, exogenous phosphorylation was stimulated by cyclic AMP. The specific activity of the membrane enzyme catalyzing the latter reaction was found to be higher than that of the enzyme present in the 100 000 × g supernatant of rat liver. The parallel effect of cyclic AMP on (Na+—K+)-ATPase and on the phosphorylation of membrane protein, and the finding that the latter reaction involves the formation of acylphosphate bonds, may be indicative of a possible role of endogenous phosphorylation in the regulation of (Na+—K+)-ATPase activity.

Increased synthesis of cyclic AMP and short-term plastic changes in the segmental ganglia of the LeechHirudo medicinalis

Summary1.The effects of different neurotransmitters on cAMP levels in segmental ganglia ofHirudo were studied. Serotonin and dopamine caused a significant increase in cAMP levels. Norepinephrine, octopamine, glutamate, and carbachol were ineffective.2.The stimulation of cAMP levels by dopamine was blocked by increasing the concentration of Mg2+ ions in the incubation bath. Serotonin was still effective in stimulating cAMP synthesis in the presence of high Mg2+ concentrations. Imidazole, a cAMP-phosphodiesterase activator, markedly inhibited the increase in cAMP induced by serotonin. Serotonin, but not dopamine, significantly stimulated adenylate cyclase activity in homogenates of segmental ganglia. The serotonin effect was blocked by RMI 12330A.3.Electrical stimulation of peripheral roots induced excitation of the fast conducting system. Repetitive stimulation (0.1/sec) resulted in a waning of firing discharge (habituation). A strong noxious stimulus applied to another pathway gave rise to a potentiation of the depressed response (dishabituation). The dishabituation was abolished by methysergide.4.These results indicate that serotonin may mediate short-term plastic changes such as dishabituation through a mechanism involving cAMP in the leechHirudo.

Electrophysiological evidence for a PGE-mediated presynaptic control of acetylcholine output in the guinea-pig superior cervical ganglion

Abstract Intracellular recordings from single ganglion neurons show that 10 −8 −10 −7 M PGE 1 reversibly blocks synaptic transmission in isolated preparations of the guinea-pig superior cervical ganglion (SCG), when added to the superfusing medium. Neither resting potential nor membrane resistance of the impaled neurons appear to be affected by PGE 1 . Quantal analyses of transmitter release demonstrate that the number of quanta liberated per volley is sharply reduced by PGE 1 treatment whereas the amplitude of the elementary event does not appear to be significantly changed.

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE1 (prostaglandin E1). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF1α was less effective and neither PGE2 nor PGF2α had any effect. Dopamine and PGE, alone or in combination, did not modify low Km cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μM), which increased cyclic AMP as much as 10 μm‐dopamine, showed no synergistic effect when tested in the presence of PGE1 or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE1 Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE1 as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE1

Heterosynaptic facilitation and behavioral sensitization are inhibited by lowering endogenous cAMP in Aplysia

An adenylate cyclase inhibitor, RMI 12330A, is able to depress cAMP synthesis stimulated by serotonin in the abdominal ganglion of Aplysia depilans and punctata. This substance reversibly blocked the heterosynaptic facilitation, induced by activation of serotonergic pathways, of the EPSP recorded from L7 motoneuron in abdominal ganglion after electrical stimulation of the siphon nerve. RMI 12330A, injected into whole unrestrained animals, inhibited the short-term dishabituation of the siphon withdrawal reflex. These findings demonstrate that the increase of endogenous cAMP in the sensory neurons mediating the gill and siphon withdrawal reflex is an essential step in the mechanism of potentiation of the transmitter output underlying heterosynaptic facilitation and short-term behavioral sensitization.

Soluble and membrane-bound adenylate cyclase activity in Yoshida ascites hepatoma

Abstract In Yoshida hepatoma cells, adenylate cyclase was found to be present in the plasma membrane as well as in the supernatant fraction. Both activities were lower than that obtained with liver membranes. During tumor growth enzyme activity decreased in the membrane and rose in the supernatant. The response of soluble and membrane-bound adenylate cyclase to epinephrine, glucagon and fluoride was markedly different. The hormone concentrations required for maximal effect were higher for the tumor than for the liver enzyme. These findings suggest that tumor cells tend to escape hormonal control.

cAMP-dependent and cAMP-independent modulation of synaptic transmission in guinea-pig superior cervical ganglion

RMI 12330A dose-dependently inhibits cAMP accumulation induced by PGE2 in guinea-pig superior cervical ganglion (SCG). Norepinephrine (NE) is unable to modify cAMP concentration in the same preparation. On the other hand, both PGE2 and NE block the firing discharge of guinea-pig SCG neurons elicited by electrical stimulation (1 Hz) of the cervical sympathetic trunk. The effect of PGE2, but not that of NE, is prevented by RMI 12330A, suggesting that NE and PGE2 act through different pathways