Cellular Cyclic GMP Levels Research Articles

Interaction of the calcium channel activating 3H-BAY K 8644 and inhibiting 3H-verapamil with specific receptor sites on cultured beating myocardial cells.

Binding properties of the calcium channel activating dihydropyridine (DHP), H-BAY K 8644, and the inhibiting 3H-verapamil were demonstrated in monolayer cultures of beating cardiac cells. 3H-BAY K 8644 specific binding was dependent on the presence of extracellular calcium, the affinity was modulated by Ca2+, but Hill coefficients remained unaffected. BAY K 8644 stimulated myocardial contractility in resting and beating myocytes. In contrast to beta-adrenoceptor agonists, however, cellular levels of cyclic AMP and cyclic GMP in cultured myocytes remained unchanged by the compound. Dihydropyridine derivatives of both the calcium channel activating BAY K 8644 as well as the Ca2+ entry blocking DHPs of the nifedipine or nimodipine type yielded very low affinity to other receptors measured in brain and heart membranes. 3H-BAY K 8644 binding sites proved to be highly specific for various potently displacing DHP derivatives and discriminated between optical isomers (stereoselectivity) with inhibition constants (Ki) in the nanomolar range. The heterogeneous shapes of the competition curves also imply interactions of these compounds with different (sub-)sites of the DHP receptor that represents one locus of interaction in regulating transmembranal Ca2+ currents. The other specific site of action for the potent diphenylalkylamines, clearly different to the DHP receptor was characterized with 3H-verapamil. The equilibrium dissociation constant, Kd in cultured myocytes ranged between 16-25 nM, and binding capacity, Bmax amounted to about 1.85 pmol/mg of protein. The different mode of competitions indicates the involvement of more than one 3H-verapamil binding site. The interrelation of the structurally heterogeneous channel modulators with the differently radiolabelled receptor (sub-)sites located in or near by the calcium channel may represent new approaches in investigating the nature of action of these potent compounds.

Effects of Calcitonin and Substance P on the Transport of Ca, Na, and Cl Across Rat Ileum in Vitro

Both salmon calcitonin (SCT) and substance P decreased ileal Na absorption, changed Cl transport from net absorption to net secretion and elevated the short circuit current when added in vitro at concentrations of 10 microng per ml to solutions bathing the serosal surface of rat ileum which had been stripped of its serosal muscle coat. The effects of substance P were of greater magnitude but shorter duration than SCT. Both peptides also increased the bidirectional fluxes of Ca but did not alter net Ca movement. The changes in Na and Cl fluxes and short circuit current are identifical to those which occur when cellular levels of cyclic AMP increased. However, incubation of ileal mucosa with SCT or substance P did not cause a detectable change in cellular levels of cyclic AMP or cyclic GMP. Both the mechanism of action and the possible physiological functions of SCT and substance P in the regulation of electrolyte transport require further investigation. The results with SCT appear to confirm prior suggestions that calcitonin may act directly to produce secretory diarrhea under pathophysiological conditions.

Increased cyclic GMP levels associated with contraction in muscle fibres of the giant barnacle

CYCLIC GMP has been implicated as an intracellular second messenger in a variety of systems. In particular, a number of hormones and neurotransmitters have been shown to raise cellular cyclic GMP levels in vitro1–9. In many of these systems, however, Ca2+ entry seems to be responsible both for the physiological effect of the hormone and for the hormone-stimulated increase in cyclic GMP (refs 4–9). We have investigated the relationship between calcium and cyclic GMP levels in muscles of the giant barnacle Balanus nubilus. This preparation has a number of advantages. Within fairly narrow limits intracellular free calcium levels in the resting muscle are known10. The depolarisation-induced increase in free calcium, and the resulting activation of contraction, have also been extensively documented10,11. In addition, the depolarisation-induced changes in intracellular free calcium can be easily manipulated because the extracellular fluid is the apparent source of the calcium which couples excitation to contraction12. We report here that either KC1 depolarisation or nerve stimulation increases the cyclic GMP content of barnacle muscle. This increase seems to be associated with Ca2+ entry rather than with a neurotransmitter–receptor interaction per se. This is the first report of an increase in cyclic GMP associated with contractile activity in cross-striated muscle.

The modulating influence of cyclic nucleotides upon lymphocyte-mediated cytotoxicity.

The capacity of allosensitized thymus-derived lymphocytes to destroy target cells bearing donor alloantigens is modulated by the cellular levels of cyclic AMP and cyclic GMP. Increases in the cyclic AMP levels of attacking lymphocytes by stimulation with prostaglandin E(1), isoproterenol, and cholera toxin inhibit lymphocyte-mediated cytotoxicity; whereas, depletion of cyclic AMP with imidazole enhances cytotoxicity. The augmentation of cytotoxicity produced by cholinergic stimulation with carbamylcholine is not associated with alterations in cyclic AMP levels and is duplicated by 8-bromo-cyclic GMP. The effects of activators of adenylate cyclase, cholinomimetic agents, and 8-bromocyclic GMP are upon the attacking and not the target cells and occur at the time of initial interaction of attacking and target cells. Indeed, the level of cyclic nucleotide (cyclic AMP and cyclic GMP) at the time of initial cell-to-cell interaction determines the extent of cytotoxicity.