Hyperpolarization- and depolarization-activated Ca2+ currents in Paramecium trigger behavioral changes and cGMP formation independently.

Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca2+ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca2+ entry. As a function of extracellular Ca2+ Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization, and formation of cGMP, i.e., the reported hyperpolarization-activated Ca2+ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling of the depolarization-activated, ciliary Ca2+ inward current and ciliary cGMP formation.Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the intracellular cGMP formation. The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral changes are triggered by smaller Ca2+ inward currents than the formation of intracellular cGMP.

Pyrrolo[2,3-d]pyrimidines as inhibitors of cAMP-phosphodiesterase. Structure-activity relationship.

The effects of pyrrolo[2,3-d]pyrimidine compounds (7-desazapurines) on cAMP hydrolyzing, calmodulin dependent and calmodulin independent phosphodiesterase were studied. Phosphodiesterase inhibition depended on the chemical nature of substituents attached to the pyrrolo-pyrimidine-nucleus at positions 2, 4, 5, 6 and 7. Among a total of 28 compounds tested, the 4-amino-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine-5,6-dicarbaldehyde (9) was the most potent inhibitor of phosphodiesterase activity (IC50 = 16 microM). In addition to the 5,6-disubstitution, position 2 of the pyrrolo-pyrimidine derivatives had to be unsubstituted and position 4 had to bear an amino-group for an optimal inhibitory effect. Calmodulin dependent and calmodulin independent isozymes were affected to the same extent. Inhibition of PDE activity was reversible upon removal of the pyrrolo-pyrimidine derivative 9 and non-competitive with respect to cAMP (Ki = 27 microM).

Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase.

Ca2+-regulated guanylate cyclase in ciliary membranes from Paramecium contained tightly bound calmodulin. Antisera against calmodulin from Tetrahymena and soybean inhibited enzyme activity. EGTA did not easily release calmodulin; however, La3+ inhibited guanylate cyclase by dissociation of calmodulin. While La could not replace Ca in the activation of guanylate cyclase, it substituted for Ca2+ in the activation of calmodulin-dependent phosphodiesterase from pig brain independently of whether homologous or Paramecium calmodulin was used. After removal of endogenous calmodulin from guanylate cyclase, reconstitution was achieved with calmodulin from Paramecium, Tetrahymena, pig brain, and soybean. Ca2+-binding proteins lacking trimethyllysine like calmodulin from Dictyostelium, parvalbumin, and troponin C failed to restore enzyme activity. The properties of the native and reconstituted guanylate cyclase/calmodulin complex were compared. Reassociation of calmodulin with its target enzyme was weak since all calmodulin remained in the supernatant after a single centrifugation. While most enzyme characteristics remained unchanged in the reconstituted complex, the inhibition by Ca greater than 100 microM was of a mixed-type compared to noncompetitive inhibition in the native enzyme. The regulation of the enzyme by cations was also altered. Whereas Ca was the most potent and specific activator of the native enzyme, in the reconstituted system Sr was far more effective.

Cyclic adenosine 3',5'-monophosphate in rat cerebral cortical slices: effects of methoxamine and clonidine.

In incubated slices of cerebral cortex from Sprague-Dawley rats, methoxamine and clonidine have no effect on basal levels of cyclic AMP. Methoxamine effectively inhibits the noradrenaline-stimulated formation of cyclic AMP. The inhibitory constant for methoxamine was 12.6 mumol/l. In the presence of 100 mumol/l adenosine, methoxamine does not inhibit the activity of noradrenaline, but is capable to activate alpha-adrenergic receptors leading to enhanced formation of cyclic AMP. The mechanism by which adenosine alters adrenergic receptors to become methoxamine-sensitive is not known. Clonidine inhibits the effect of noradrenaline alone or in combination with adenosine on the cyclic-AMP-generating system. It does not, as reported earlier, enhance the activity of submaximal concentrations of the beta-adrenergic agonist isoproterenol. These data do not support the concept of adrenergic receptors which require both, alpha- and beta-stimulation for maximal activation of adenylate cyclase.

Adenylate cyclase and phosphodiesterase from brain tissue: different stabilities during incubation of cerebral cortical slices.

Adenylate cyclase and phosphodiesterase were prepared from brain slices from guinea-pig. The specific activity of adenylate cyclase declined rapidly with increasing incubation time of tissue slices, while phosphodiesterase activity was almost uneffected by the incubation of brain slices.

Stimulation of adenosine 3',5'-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium.

In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical alpha-type receptor. 5-Hydroxytryptamine, prostaglandin E1 and dopamine do not significnatly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K+ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.