Mutations affecting the delayed rectifier potassium current in Drosophila.

Mutations and pharmacological agents have been used to resolve and analyze several K(+) currents in Drosophila. Mutations that affect channels carrying the voltage-activated I(A) and the Ca(2+) -activated I(CF) have helped greatly in analyzing the structure, function and regulation of these channels. We now report mutations that selectively affect the delayed rectifier current, I(K). Flies mutagenized with ethylmethanesulfonate were screened for temperature-induced paralysis. Paralytic mutants identified in the screen were examined for K(+) currents in the larval body-wall muscles. The z66 mutant larvae showed a significant reduction in I(K). The mutation did not affect other K(+) currents (I(A), I(CF), or I(CS) ) or the Ca(2+) channel current in the muscles. Another mutation, z4, which showed reduced I(K), failed to complement z66. Genetic analysis localized the gene disrupted by z66 and z4 to the left arm of chromosome 3, in the 63A1-63B6 region on polytene chromosomes. The z66 and the z4 mutations, which lie in the Shab K(+) channel gene, provide an opportunity to undertake analysis of the functioning of these channels and to study the role of these channels in membrane excitability.

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.

Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.

Modulation of the dihydropyridine-sensitive calcium channels in Drosophila by a phospholipase C-mediated pathway.

Disruption of phospholipase C-beta (PLC) by the norpA mutations of Drosophila renders flies blind by affecting the light-evoked photoreceptor potential. We report here that the norpA-coded PLC modulates the 1,4-dihydropyridine (DHP)-sensitive Ca2+ channels in larval muscles. The DHP-sensitive current was reduced in the norpA mutants. Application of 1 microM phorbol 12-myristate 13-acetate (TPA) and 1 microM phorbol 12,13-didecanoate (PDD), activators of protein kinase C (PKC), rescued the current in the mutant fibers without significantly affecting the normal current. 4Alpha-phorbol 12,13-didecanoate (4alphaPDD), an inactive analog of PDD, did not affect either the normal or the mutant current. One micromolar bisindolylmaleimide (BIM), an inhibitor of PKC, reduced the current in the normal fibers without affecting the mutant current. 300 microM sn-1,2-dioctanoyl-glycerol (DOG), an analog of diacylglycerol (DAG), increased the current in the mutant fibers. These experiments suggest that the DHP-sensitive Ca2+ channels in Drosophila may be modulated by the PLC-DAG-PKC pathway, and that the same PLC isozyme which is involved in phototransduction in the adult flies may also modulate muscle Ca2+ channels in the larval stage of development.

The effects of cocaine injections on mouse thymocyte population.

C57 BL mice were injected daily with either saline or varied doses of cocaine (5-50 mg/kg), and thymocyte subpopulations were analyzed 4 hr after the fifth injection. Mice injected with either 25 or 50 mg/kg of cocaine showed a decrease in the percentage of CD4+8+ cells and increase of CD4-8-, CD4+, and CD8+ cells. The absolute numbers of each subpopulation, calculated by multiplying the percentage of each subpopulation with the total cell number, revealed an extensive decline in CD4+8+, a decrease in CD8+, an increase in CD4-8-, and no change in the CD4+ subpopulation. Flow cytometric analysis of thymocytes and electrophoresis of the thymocyte DNA revealed a dosage-dependent increase in cells undergoing programed cell death with apoptosis. Culturing of thymocytes from control or drug-treated mice demonstrated an inverse relationship between cell viability and cocaine concentrations, suggesting that in vivo cocaine, or its biological products, may damage thymocytes. Incubation of normal cells with cocaine showed a dose-dependent decrease of viability with identical patterns of the alteration of cell subpopulations observed in vivo. A dose-dependent increase of apoptosis was also observed. In summary, we demonstrate a selective in vivo cocaine-induced alteration of the thymocyte subpopulations and identified programed cell death with apoptosis as the likely mechanism mediating this thymic atrophy. The comparable findings observed in vivo and in vitro support the concept that cocaine may directly affect some features of thymocyte biology, and suggest the usefulness of the in vitro system in studying cocaine effects on thymocyte biology.

Pharmacological analysis of heartbeat in Drosophila.

Analysis of the mechanisms underlying cardiac excitability can be facilitated greatly by mutations that disrupt ion channels and receptors involved in this excitability. With an extensive repertoire of such mutations, Drosophila provides the best available genetic model for these studies. However, the use of Drosophila for this purpose has been severely handicapped by lack of a suitable preparation of heart and a complete lack of knowledge about the ionic currents that underlie its excitability. We describe a simple preparation to measure heartbeat in Drosophila. This preparation was used to ask if heartbeat in Drosophila is myogenic in origin, and to determine the types of ion channels involved in influencing the heart rate. Tetrodotoxin, even at a high concentration of 40 microM, did not affect heart rate, indicating that heartbeat may be myogenic in origin and that it may not be determined by Na+ channels. Heart rate was affected by PN200-110, verapamil, and diltiazem, which block vertebrate L-type Ca2+ channels. Thus, L-type channels, which contribute to the prolonged plateau of action potentials in vertebrate heart, may play a role in Drosophila cardiac excitability. It also suggests that Drosophila heart is subject to a similar intervention by organic Ca2+ channel blockers as the vertebrate heart. A role for K+ currents in the function of Drosophila heart was suggested by an effect of tetraethylammonium, which blocks all the four identified K+ currents in the larval body wall muscles, and quinidine, which blocks the delayed rectifier K+ current in these muscles. The preparation described here also provides an extremely simple method for identifying mutations that affect heart rate. Such mutations and pharmacological agents will be very useful for analyzing molecular components of cardiac excitability in Drosophila.

Resolution and pharmacological analysis of the voltage-dependent calcium channels of Drosophila larval muscles.

Voltage-dependent calcium channels play a role in many cellular phenomena. Very little is known about Ca2+ channels in Drosophila, especially those in muscles. Existing literature on neuronal Ca2+ channels of Drosophila suggests that their pharmacology may be distinct from that of vertebrate Ca2+ channels. This raises questions on the pharmacology and diversity of Ca2+ channels in Drosophila muscles. Here we show that the Ca2+ channel current in the body-wall muscles of Drosophila larvae consists of two main components. One component is sensitive to 1,4-dihydropyridines and diltiazem, which block vertebrate L-type Ca2+ channels. The second component is sensitive to amiloride, which blocks vertebrate T-type Ca2+ channels. In contrast to Drosophila brain membrane preparations in which a majority of the Ca2+ channels are phenylalkylamine-sensitive but dihydropyridine-insensitive, the major current in the muscles was dihydropyridine-sensitive but relatively less sensitive to verapamil. This might indicate an underlying tissue specific distribution of distinct subtypes of dihydropyridine/phenylalkylamine-sensitive Ca2+ channels in Drosophila. Low verapamil sensitivity of the dihydropyridine-sensitive current of Drosophila muscles also set it apart from the vertebrate L-type channels which are sensitive to 1,4-dihydropyridines, benzothiazepines as well as phenylalkylamines. The dihydropyridine-sensitive current in Drosophila muscles activated in a similar voltage range as the vertebrate L-type current. As with the vertebrate current, blockade by dihydropyridines was voltage dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

[Anti-ulcer effect of anisodamine in rats].

Anisodamine, an analog of atropine, was isolated first in China. Anisodamine 12.5, 25, 50 mg.kg-1 ig protected the gastric mucosal damage induced by indomethacin, restraint, pyloric ligation or absolute ethanol in rats. Pretreatment with anisodamine 25 mg.kg-1 ig bid for 3 d decreased the incidence of ulcers in all of these models. The inhibitory rates were 27.4%, 75.9%, 80% and 65.4%, respectively. The secretion and activity of pepsin, the content of hexosamine in gastric tissue, the production of malondialdehyde and activity of superoxide dismutase were not affected by anisodamine except the secretion of gastric acid. It increased output of basal bicarbonate from 0.255 +/- 0.01 to 0.285 +/- 0.01 mumol.min-1 (P less than 0.05) and pH of content of stomach from 4.3 +/- 0.3 to 5.1 +/- 0.5 (P less than 0.05) when given by anisodamine 20 mg.kg-1 ig. It is apparent that both the inhibition on gastric acid secretion and augmentation of basal bicarbonate content of stomach are related to anti-ulcer effect of anisodamine.

[Antiulcer action and mechanism of trifluoperazine in rat stomach].

Trifluoperazine (TFP) 5, 10, 20 mg.kg-1 ig inhibited the formation of gastric ulcers induced by pyloric ligation, stress and indomethacin in rats and showed dose-effect dependence. TFP 10, 20 mg.kg-1 ig depressed the secretion of gastric juice, acid, and pepsin, but TFP 5, 10, 20 mg.kg-1 ig had no influence on the pepsin activity. TFP 20 mg.kg-1 ig inhibited the gastric H+, K(+)-ATPase activity of both stress and indomethacin ulcers in rats in vivo, and the gastric H+, K(+)-ATPase activity was also inhibited by TFP 50 mumol.L-1 in vitro. The results suggested that the inhibition of gastric H+, K(+)-ATPase activity and gastric secretion might be related to the antiulcer mechanism of TFP.

[Anti-gastric ulcer activity of zinc sulfadiazine in rats].

Zinc sulfadiazine (ZnSD) 50, 100, 200 mg/kg ig inhibited the formation of gastric ulcer induced by indomethacin, stress and pyloric ligation in rats respectively and showed dose-dependently. ZnSD 200 mg/kg ig accelerated the healing of gastric ulcer induced by acetic acid. ZnSD 25 mg/kg ig was effective in preventing ethanol-induced damage of rat gastric mucosa. The amount of gastric mucus glycoprotein in gastric tissues was increased by ZnSD. In general, ZnSD did not influence the volume of gastric juice and pepsin output, but ZnSD 200 mg/kg ig decreased gastric acidity. In vitro, ZnSD also influenced the neutralization of acid. It is suggested that antiulcer action of ZnSD may be related to its preservation of the gastric mucosal barrier and neutralization of acid.

[Anti-ulcer activity of Na-gamma-hydroxybutyrate in rat stomach].

The antigastric ulcer activity of Na-gamma-hydroxybutyrate (GHBA) was studied on various experimental ulcers in rats. The results showed that the number of gastric ulcers induced by sc indomethacin 20 mg/kg, restraint stress or pyloric ligation were markedly diminished by GHBA. Pretreatment with GHBA 10 mg/kg im bid 5 times revealed significant anti-ulcer activity in all of these models. The inhibitory rates of ulcer were 42, 71 and 51% for indomethacin, restraint stress and pyloric ligation, respectively. Chronic gastric ulcer induced by 10% acetic acid was also inhibited by GHBA 10 mg/kg im bid for 8 days. The ulcer inhibitory rate was 73%. The amount of gastric mucus glycoprotein and [3H] TdR incorporation into gastric tissues was increased by GHBA, but the volume of gastric juice, and acid and pepsin output were not influenced. It is suggested that the prevention of GHBA on gastric mucosal damage may be related to its preservation of the gastric mucosal barrier.