Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.     

The cytotoxic effects of estradiol-17 beta, catecholestradiols and methoxyestradiols on dividing MCF-7 and HeLa cells

In this study the cytotoxic effects of high concentrations (greater than or equal to 1 x 10(-6) M) of estradiol-17 beta (E2), 2-/4-hydroxyestradiol-17 beta (2-/4-OHE2) and 2-/3-/4-methoxyestradiol-17 beta (2-/3-/4-MeOE2) were determined on dividing MCF-7 and HeLa cells. The 2-MeOE2 metabolite followed by 2-OHE2 and E2 (in this order) proved to be extremely toxic to dividing MCF-7 and HeLa cells. The cytotoxic effect on these cells comprised uneven chromosome distribution. Indirect immunofluorescent studies, in which monoclonal anti-alpha-tubulin antibodies were used, showed that these compounds (2-MeOE2 greater than 2-OHE2 greater than E2) at high concentrations caused abnormal and fragmented polar formations as well as disorientated microtubule arrangement in the dividing MCF-7 and HeLa cells. The 4-OHE2 and 3-/4-MeOE2 metabolites had little or no cytotoxic effects on dividing cells. The large number of abnormal metaphases seen in HeLa cells exposed to 2-MeOE2 suggested that this metabolite may be the ultimate cytotoxic compound. The reduction in the number of HeLa cells with abnormal metaphase configurations after exposure to 2-OHE2 plus quinalizarin (an inhibitor of catechol-O-methyltransferase) indicated that the production of 2-MeOE2 is necessary for the formation of abnormal spindles in metaphase. Quinalizarin treatment in the presence of 2-MeOE2 had no effect on the large number of abnormal metaphases. We therefore conclude that neither E2 nor 2-OHE2, but a high concentration of 2-MeOE2 is responsible for abnormal spindle formation. In additional experiments the number of normal and abnormal dividing HeLa cells were greatly reduced when simultaneously exposed to E2 and 2-/4-hydroxylase-inhibitor alpha-naphthoflavone.     

Effect of indomethacin on airway contraction and the release of LTC4-like material

Ovalbumin (OA) and arachidonic acid (AA) were used to induce contractions of sensitized guinea-pig tracheal and lung preparations in the presence and absence of indomethacin. Leukotriene (LT)C4-like material released from these tissues was extracted from the bathing fluid and measured by radioimmunoassay. Challenge with either OA or AA induced release of LTC4-like material from both parenchyma and trachea, AA inducing a greater release than OA although OA induced greater contractions. This suggested that OA-induced the synthesis of other bronchoconstrictor compounds than LTC4. Although indomethacin enhanced OA- and AA-induced contractions of trachea, there was no enhancement of the release of LTC4-like material, suggesting enhancement by indomethacin was a result of the inhibition of the synthesis of prostaglandin E2 and not diversion of AA into the lipoxygenase pathway. Indomethacin had no effect on OA-induced contractions of parenchyma, but attenuated those induced by AA. Indomethacin had no modulatory effect on the release of LTC4-like material in the parenchyma. The results demonstrate that indomethacin does not result in increased synthesis of LTs in the airways.     

The ionic requirements for regulation by molluscan thin filaments

Initial studies on molluscan muscle regulation indicated that thin filaments do not confer Ca2+-dependence on vertebrate myosin ATPase, and hence that molluscan muscles do not possess thin filament-linked regulatory systems. Subsequently it was shown that molluscan thin filaments do, in fact, impart Ca2+-sensitivity but only at Mg2+ concentrations greater than those used in the earlier studies. In the present study it is shown that Mg2+ prevents significant dissociation of tropomyosin and troponin subunits from thin filaments at the low monovalent ion concentrations typically employed to assay actomyosin ATPase; as a result Mg2+ allows expression of the molluscan thin filament regulatory system under these conditions.     

Potent antagonistic action of synthetic analogues of APGWGNamide,an antagonist of molluscan neuropeptide APGWamide

Fifty-five kinds of analogues of APGWGNamide (Ala-Pro-Gly-Trp-Gly-Asn-NH2), which is an antagonist of molluscan neuropeptide APGWamide, were synthesized and their antagonistic activities were examined on two molluscan smooth muscles. Among all the analogues tested, on spontaneous contraction of the crop of the land snail, Euhadra congenita, APGWG(L-biphenylalanine, Bip)amide showed the most potent antagonistic activity and its potency was 50-100 times higher than that of APGWGNamide. Likewise, on phasic contraction of the anterior byssus retractor muscle (ABRM) of the sea mussel, Mytilus edulis, the effect of APGWG(D-homophenylalanine, dHfe) was the most potent and showed 5-10 times stronger activity than that of APGWGNamide. In the tolerance test to known exo- and endopeptidases or the crop tissue homogenate, APGWGNamide was not only easily degraded by a proline-specific endopeptidase but also by the homogenate. Two kinds of potent antagonists were thus developed: APGWG(Bip)amide and APGWG(dHfe)amide, which will be useful tools for investigation of the function of APGWamide in the snail and the mussel, respectively.     

Toxicity of 1-methyl-4-phenylpyridinium derivatives in Escherichia coli.

Several derivatives of 1-methyl-4-phenylpyridinium (MPP+), i.e., 1-methyl-4-(4'-nitrophenyl)pyridinium (1), 1-methyl-4-(4'-cyanophenyl)pyridinium (2), 1-methyl-4-(3'-nitrophenyl)pyridinium (3), 1-methyl-4-(4'-chlorophenyl)pyridinium (4), 1-methyl-4-(4'-acetamidophenyl)pyridinium (5), and 1-methyl-4-(4'-aminophenyl)pyridinium (6), were synthesized in order to compare their toxicity with that of paraquat (PQ2+) in Escherichia coli. Addition of compounds 1, 2, and 3 to aerobic E. coli cell suspensions caused extracellular ferricytochrome c reduction, which was inhibited by superoxide dismutase in the same manner as that in the case of PQ2+. The rate of the ferricytochrome c (cyt. c) reduction was in the order of PQ2+ greater than 1 greater than 2 greater than 3, which is the same as that of the redox potentials of these compounds. On the other hand, MPP+, 4, 5, and 6, which have more negative potentials, had no effect on the cyt. c reduction. Compound 1 inhibited the growth of E. coli under aerobic conditions, but not under anaerobic conditions. The results show that compound 1 can act as a mediator for production of superoxide (O2-.), which seriously injures E. coli cells. However, though compounds 2 and 3 catalyzed the production of O2-. in E. coli cells, their activity of O2-. production was much lower than that of compound 1 or PQ2+. Thus, compound 3 had no effect on growth or survival of E. coli at 1 mM, while compounds 2 and 4 had both bacteriostatic and bacteriocidal effects which were independent of dioxygen (O2). The results show that the toxic mechanism is different from that of compound 1. MPP+, 5, and 6 had no effect on growth of E. coli. This paper shows that compound 1 is a novel enhancer of intracellular superoxide production, though the mechanism of toxicity of compounds 2 and 4 is not clear yet. The results suggest that the redox potential is a crucial factor for manifestation of the activity.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Central action of tachykinins on activity of expiratory pumping muscles.

The central effects of tachykinins (substance P, neurokinin A, and neurokinin B) on the distribution of the motor activity to rib cage and abdominal expiratory muscles were studied in anesthetized tracheotomized spontaneously breathing dogs and cats. Intracisternal application of substance P (11 dogs) in doses of 10(-5) to 10(-4) M caused diaphragm electrical activity to change insignificantly from 19.3 +/- 1.9 to 24.8 +/- 3.2 units (P greater than 0.05), produced a moderate increase of triangularis sterni activity from 12.6 +/- 2.2 to 19.2 +/- 2.2 units (P less than 0.05), and stimulated a large increase of transversus abdominis activity from 9.4 +/- 2.7 to 28.5 +/- 2.6 units (P less than 0.01). Comparable effects were seen with similar doses of neurokinin A (8 dogs) and neurokinin B (3 dogs) administered intracisternally. Local application of substance P to the ventral medullary surface (5 dogs and 4 cats) also caused expiratory muscle activity to increase more than diaphragm activity, and in addition transversus abdominis activity increased to a larger extent than triangularis sterni activity. Furthermore, administration of the substance P antagonist [D-Pro2,D-Trp7,9]-SP to the ventral medullary surface decreased respiratory motor output, with expiratory muscles activity being attenuated to a greater extent than diaphragm activity. Application of neurotensin and N-methyl-D-asparate to the ventral surface of the medulla produced responses similar to those observed as a result of central administration of tachykinin peptides. The results suggest that 1) mammalian tachykinins are involved in the regulation of thoracic and abdominal expiratory muscle activity, 2) these muscles manifest substantial differences in their electrical responses to excitatory neuropeptides acting centrally, and 3) inputs from modulatory neurons located in this vicinity of the ventral medullary surface seem to be distributed unevenly to different expiratory premotor and/or motoneurons.     

Preparation of an 125I-labeled red pigment-concentrating hormone analog.

The red pigment-concentrating hormone (RPCH) from crustacea is an octapeptide with the following structure: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH₂, [4-Tyr]RPCH has been synthesized and was 4 times as active as RPCH in the Leander assay. Iodination of the hormone analog using chloramin-T, lactoperoxidase, solid-state lactoperoxidase, and thallium chloride was carried out and the results were compared. ¹²⁵I-labeled [4-Tyr]RPCH was prepared using a modificated chloramin-T method and was purified from unlabeled and diiodinated peptide by chromatography on Sephadex LH-20.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Effect of the diaminocyclohexane carrier ligand on platinum adduct formation, repair, and lethality

Platinum compounds with the diaminocyclohexane (dach) carrier ligand are of particular interest because cell lines that have developed resistance to platinum compounds in general often retain sensitivity to dach-platinum compounds, suggesting that the dach carrier ligand affects the formation, repair, or lethality of platinum-DNA adducts. The effect of the dach ligand on platinum adduct formation was assessed by using the (HaeIII-HindIII)146 fragment of pBR322 treated to give equal amounts of dach- or ethylene-diamine-platinum adducts. The sites of adduct formation were mapped by digestion with Escherichia coli ABC excinuclease. There were no significant effects of the dach carrier ligand on the types or sites of platinum adduct formation. The effect of the dach ligand on platinum adduct repair was determined by using synthetic oligomers designed to have single, specific platinum adducts (G monoadduct; GG, AG, or GNG diadduct) with either the dach or ethylenediamine (en) carrier ligand. These adducts differed significantly in their ability to serve as substrates for ABC excinuclease with GNG greater than or equal to G greater than AG greater than GG. The dach carrier ligand had little effect on the recognition of AG and GG adducts by ABC excinuclease, but significantly improved the ability of ABC excinuclease to excise G monoadducts and GNG diadducts. These data suggest that if the carrier ligand has any effect on the repair of platinum adducts, it is more likely to exert that effect on the repair of platinum monoadducts or GNG diadducts rather than on the more abundant AG or GG diadducts. [14C]Thiourea incorporation was used to quantitate the rate of monoadduct to diadduct conversion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on a modulatory role for adenosine in antigen and arachidonic acid induced contractions of guinea pig airways

The modulatory effects of adenosine and selected derivatives were examined on antigen and arachidonic acid (AA) induced contractions of indomethacin-treated tracheal spirals and lung parenchymal strips from actively sensitized guinea pigs. Adenosine (up to 2 X 10(-4) M) had no effect on antigen-induced contractions, but inhibited AA-induced contractions by 30-40% if added 30 min prior to challenge. The weak effect of adenosine suggests that endogenous adenosine may only have a limited modulatory role in allergic bronchospasm. 2-Chloroadenosine (10(-6)-10(-4) M) dose-dependently inhibited antigen- and AA-induced contractions of trachea, but was considerably less effective on parenchyma. The substituted adenosine derivatives, R-phenylisopropyladenosine (R-PIA) and 5'-(N-ethylcarboxamido)-adenosine (NECA), and the adenosine transport inhibitor, 6-[p-nitrobenzyl)thio]-9-beta-D-ribofuranosyl purine, were also active as modulators, but their activity was relatively weak and varied with the stimulus and the tissue. An order of potency for R-PIA, NECA, and 2-chloroadenosine could not be determined and 8-phenyltheophylline (10(-5) M) was not an effective inhibitor of the effects of adenosine or the adenosine derivatives. This suggests that adenosine and its derivatives may modulate cells through mechanisms other than activation of conventional A1 and A2 receptors. A lack of specificity for the adenosine derivatives must also be considered.     

A possible molecular ancestor for mollusk APGWamide,insect adipokinetic hormone,and crustacean red pigment concentrating hormone

Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar α helix–random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families. The APGWamide genes are larger than the AKH/RPCH genes. The sequence environment occupied by introns is similar in AKH/RPCH and APGWamide genes. Such similarities suggest that these peptide families might have been originated by gene rearrangements from a common ancestor having either an AKH/RPCH/APGWamide-like structure or both an AKH/RPCH-like and an APGWamide-like structures. In the former model, DNA fragments could have been gained when the ancestor evolved to mollusks and it could have lost nucleotides when the progression to mandibular arthropods took place. In the second model, AKH/RPCH-like structures could have been fused during evolution toward mandibular arthropods, whereas in mollusks they could have been lost with the possible amplification of the APGWamide-like structure. Loss of domains in exon 1 may have originated the signal peptide and the first codon of the active RPCH. In exon 2, loss of domains possibly determined the junctions of codons 2 to 5 with the loss of a APGWamide copy; exon 3 underwent fewer variations. The similarity of the mollusk APGWamide precursors is closer to that of the RPCH family than the insect AKH family, indicating an earlier evolutionary departure.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Structure-function studies on red pigment-concentrating hormone, II. The significance of the C-terminal tryptophan amide

The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH.     

Inhibition of auxin transport by isoquinolinedione herbicides

2-(p-carbethoxyphenyl)-1,3(2H,4H)-isoquinolinedione (CEPIQ), an experimental herbicide, caused effects on geotropism, which are often indicative of an effect on auxin transport, in a whole plant herbicidal screen. However, it showed little or no activity in an in vitro binding assay in corn coleoptiles for the auxin-transport inhibitor,N-1-naphthylphthalamic acid (NPA). Other active isoquinolinedione analogues of this compound did, however, exhibit significant in vitro activity. Direct measurements of auxin transport in corn coleoptiles were undertaken in an attempt to resolve the apparent discrepancy between herbicidal and binding activities. In all cases examined, compounds that were highly active on whole plants were good inhibitors of auxin transport, and compounds that were weak as herbicides showed little or no effect on auxin transport. Therefore, it is concluded that the mode of action of these isoquinolinedione herbicides is the inhibition of auxin transport. Ring-opened analogues of several isoquinolinediones were synthesized and assayed in both the transport and binding assays, in order to test whether compounds in this class express their herbicidal activity by undergoing ring-opening in vivo, yielding products that are more straightforward analogues of NPA with free carboxyl groups. The homophthalamic acids had little or no activity in both assays. On the other hand, thep-ethyl- andp-ethoxy-phenyl phthalamic acids showed auxin transport inhibition comparable to the parent isoquinolinediones, but with markedly increased binding activity. These results support the possible role of ring-opening in the generation of biological activity. However, thep-carbethoxyphenyl phthalamic acid, analogous to CEPIQ, was very weak in both assays. Thus, ring-opening in vivo cannot alone account for the biological activity of this class of compounds.