Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Requirement for the action of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus

The role of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus L. was investigated. The seeds readily germinated in water and darkness at 24°C. Application of ethylene or of its precursor I-aminocyclopropane-I-carboxylic acid (ACC) slightly increased the rate of germination. Both compounds effectively antagonized osmotic inhibition by polyethyleneglycol. Application of aminoethoxyvinylglycine (AVG) reduced ethylene production by 90% but did not inhibit germination. However, germination was inhibited by 2,5-norbornadiene, a competitive inhibitor of ethylene action. This inhibition was counteracted by ethylene, ethephon or ACC and enforced by AVG. It is concluded that the action of endogenous ethylene is an indispensable factor during germination of non-dormant seeds of A. caudatus. Ethylene action is required from the start of imbibition on. In water, low levels of endogenous ethylene are sufficient for this action. PEG increased the ethylene requirement considerably.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.     

Macrophage mediation of the inhibitory effects of elevated intracellular levels of adenosine-3':5' cyclic monophosphate (cAMP) on macrophage-Trypanosoma cruzi association

Presence of theophylline and dibutyryl-cAMP—agents which cause elevation of intracellular levels of cAMP—in in vitro systems in which murine macrophages interact with virulent blood forms of Trypanosoma cruzi resulted in a marked inhibition of cell-parasite association (i.e., decreased surface binding and/or internalization). This effect was evidenced in terms of significant reductions in both the percentage of infected macrophages and the average number of trypanosomes per 100 macrophages. Pretreatment of the macrophages with these agents produced a similar inhibition whereas pretreatment of the parasites had no significant consequence on the interaction. The inhibitory effect was transient since it was no longer seen after 30 min of incubation of the treated macrophages in fresh medium. Thus, the inhibitory effect is exerted through a transient effect on the macrophage.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.