Big and slow: phylogenetic estimates of molecular evolution in baleen whales (suborder mysticeti).

Baleen whales are the largest animals that have ever lived. To develop an improved estimation of substitution rate for nuclear and mitochondrial DNA for this taxon, we implemented a relaxed-clock phylogenetic approach using three fossil calibration dates: the divergence between odontocetes and mysticetes approximately 34 million years ago (Ma), between the balaenids and balaenopterids approximately 28 Ma, and the time to most recent common ancestor within the Balaenopteridae approximately 12 Ma. We examined seven mitochondrial genomes, a large number of mitochondrial control region sequences (219 haplotypes for 465 bp) and nine nuclear introns representing five species of whales, within which multiple species-specific alleles were sequenced to account for within-species diversity (1-15 for each locus). The total data set represents >1.65 Mbp of mitogenome and nuclear genomic sequence. The estimated substitution rate for the humpback whale control region (3.9%/million years, My) was higher than previous estimates for baleen whales but slow relative to other mammal species with similar generation times (e.g., human-chimp mean rate > 20%/My). The mitogenomic third codon position rate was also slow relative to other mammals (mean estimate 1%/My compared with a mammalian average of 9.8%/My for the cytochrome b gene). The mean nuclear genomic substitution rate (0.05%/My) was substantially slower than average synonymous estimates for other mammals (0.21-0.37%/My across a range of studies). The nuclear and mitogenome rate estimates for baleen whales were thus roughly consistent with an 8- to 10-fold slowing due to a combination of large body size and long generation times. Surprisingly, despite the large data set of nuclear intron sequences, there was only weak and conflicting support for alternate hypotheses about the phylogeny of balaenopterid whales, suggesting that interspecies introgressions or a rapid radiation has obscured species relationships in the nuclear genome.

A pertussis toxin-sensitive 8-lipoxygenase pathway is activated by a nicotinic acetylcholine receptor in aplysia neurons.

Acetylcholine (ACh) activates two types of chloride conductances in Aplysia neurons that can be distinguished by their kinetics and pharmacology. One is a rapidly desensitizing current that is blocked by alpha-conotoxin-ImI and the other is a sustained current that is insensitive to the toxin. These currents are differentially expressed in Aplysia neurons. We report here that neurons that respond to ACh with a sustained chloride conductance also generate 8-lipoxygenase metabolites. The sustained chloride conductance and the activation of 8-lipoxygenase have similar pharmacological profiles. Both are stimulated by suberyldicholine and nicotine, and both are inhibited by alpha-bungarotoxin. Like the sustained chloride conductance, the activation of 8-lipoxygenase is not blocked by alpha-conotoxin-ImI. In spite of the similarities between the metabolic and electrophysiological responses, the generation of 8-lipoxygenase metabolites does not appear to depend on the ion current since an influx of chloride ions is neither necessary nor sufficient for the formation of the lipid metabolites. In addition, the application of pertussis toxin blocked the ACh-activated release of arachidonic acid and the subsequent production of 8-lipoxygenase metabolites, yet the ACh-induced activation of the chloride conductance is not dependent on a G protein. Our results are consistent with the idea that the nicotinic ACh receptor that activates the sustained chloride conductance can, independent of the chloride ion influx, initiate lipid messenger synthesis.

Heteroplasmy of mitochondrial DNA in the ophiuroid Astrobrachion constrictum.

We demonstrate the presence of mitochondrial heteroplasmy for the cytochrome oxidase I (COI) gene of the brittle star (Astrobrachion constrictum). One of the 117 individuals analyzed contained two distinct single-strand conformation polymorphism (SSCP) haplotypes differing by two substitutions; another showed sequence evidence for heteroplasmy. We used polymerase chain reaction (PCR) cloning, SSCP, and sequencing of a 480 bp region of the 5' end of COI to isolate and characterize these haplotypes. This is the first properly substantiated case of heteroplasmy in an echinoderm species and may have arisen from paternal leakage.

Identification of an 8-lipoxygenase pathway in nervous tissue of Aplysia californica.

Arachidonic acid is converted to (8R)-hydroperoxyeicosa-5,9,11, 14-tetraenoic acid (8-HPETE) during incubations with homogenates of the central nervous system of the marine mollusc, Aplysia californica. 8-HPETE can be reduced to the corresponding hydroxy acid or be enzymatically converted to a newly identified metabolite, 8-ketoeicosa-5,9,11,14-tetraenoic acid (8-KETE). These metabolites were identified by high performance liquid chromatography, UV absorbance, and gas chromatography/mass spectrometry. Stereochemical analysis of the products demonstrate that the neuronal enzyme is an (8R)-lipoxygenase. Previously we have shown that the neurotransmitters, histamine and Phe-Met-Arg-Phe-amide, activate 12-lipoxygenase metabolism in isolated identified Aplysia neurons. We now show that acetylcholine activates the (8R)-lipoxygenase pathway within intact nerve cells. Thus, both (12S)- and (8R)-lipoxygenase co-exist in intact Aplysia nervous tissue but are differentially activated by several neurotransmitters. The precise physiological role of the 8-lipoxygenase products is currently under investigation, but by analogy to the well-described 12-lipoxygenase pathway, we suggest that (8R)-HPETE and 8-KETE may serve as second messengers in Aplysia cholinoceptive neurons.

Stereochemistry of the Aplysia neuronal 12-lipoxygenase: specific potentiation of FMRFamide action by 12(S)-HPETE.

Nervous tissue of the marine mollusc, Aplysia californica, generates arachidonic acid metabolites in response to neurotransmitters such as histamine or FMRFamide. In addition, identified neurons of Aplysia respond to the pharmacologic application of some of these products, particularly those of the 12-lipoxygenase pathway. We investigated the chirality of the initial Aplysia 12-lipoxygenase product, 12-HPETE, in preparation for more detailed metabolic studies and for the analysis of the physiological activity of the endogenous lipid. Neural homogenates and intact ganglia exclusively generate 12(S)-HPETE as do the better characterized mammalian lipoxygenases. The direct application of 12(S)-HPETE to cultured sensory neurons induced a hyperpolarization which averaged 2.6 mV. We did not find any difference between the response to the naturally-occurring 12(S)-HPETE and its diastereomer, 12(R)-HPETE which is not generated in Aplysia. Both isomers were significantly more effective than 15(S)-HPETE. In contrast, 12(S)-HPETE, but not 12(R)-HPETE, was a potent modulator of the action of the molluscan neuropeptide, FMRFamide. Prior application of 12(S)-HPETE to cultured sensory neurons increased the subsequent response to a submaximal dose of FMRFamide by 60%. On the other hand, 12(R)-HPETE reduced the subsequent response to the peptide by 30%. The lack of stereospecificity in the direct effect of the lipids differs markedly from their stereospecific effects as modulators of FMRFamide action. This suggests that there may be an important neurophysiologic role for these lipid modulators which is distinct from their direct effects, and also indicates that there are multiple sites and mechanisms by which lipid hydroperoxides act on neurons in Aplysia.

Aplysia californica contains a novel 12-lipoxygenase which generates biologically active products from arachidonic acid.

Physiologic stimulation of identified neurons in ganglia of the marine mollusk, Aplysia californica, leads to the generation of arachidonic acid metabolites. Using various preparations of Aplysia nervous tissue, we have identified 12-lipoxygenase products including the inactive 12-hydroxyeicosatetraenoic acid (12-HETE) and the biologically active 12-ketoeicosatetraenoic acid (12-KETE) and 8-hydroxy-11(12)-epoxyeicosatrienoic acid (8-HEpETE). Each of these metabolites can be derived from the intermediate 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can itself activate several identified neurons in Aplysia. In spite of conflicting results in studies of mammalian brain 12-lipoxygenase, Aplysia nervous tissue clearly contains an enzymatic activity which generates stereochemically pure 12(S)-HETE. This activity is destroyed by boiling and is sensitive to nonspecific lipoxygenase inhibitors but not to agents specific for other lipoxygenases or the cyclooxygenase enzyme. The Aplysia 12-lipoxygenase is highly enriched in neural tissue and is almost completely absent in the neural sheath, which is composed primarily of connective tissue and muscle. Preliminary purification has shown that, in contrast to the previously characterized 12-lipoxygenases, the Aplysia enzyme is associated with membrane fractions and is not found in the cytosol. Further studies are in progress to determine the kinetic properties and to define the cellular and subcellular distribution of this novel lipoxygenase.

CGS 19755, a selective and competitive N-methyl-D-aspartate-type excitatory amino acid receptor antagonist.

CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) was found to be a potent, stereospecific inhibitor of N-methyl-D-aspartate (NMDA)-evoked, but not KCl-evoked, [3H] acetylcholine release from slices of the rat striatum. The concentration-response curve to NMDA was shifted to the right by CGS 19755 (pA2 = 5.94), suggesting a competitive interaction with NMDA-type receptors. CGS 19755 inhibited the binding of [3H]-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid to NMDA-type receptors with an IC50 of 50 nM, making it the most potent NMDA-type receptor antagonist reported to date. CGS 19755 failed to interact with 23 other receptor types as assessed by receptor binding, including the quisqualate- and kainate-type excitatory amino acid receptors. In crude P2 fractions, no evidence was obtained to suggest that CGS 19755 is taken up by an active transport system. Furthermore, CGS 19755 failed to affect the uptake of L-[3H]glutamate, or to interact with aconitine-induced inhibition of L-[3H]glutamate uptake, the latter finding suggesting a lack of membrane-stabilizing or local anesthetic properties. CGS 19755 selectively antagonized the excitatory effect of iontophoretically applied NMDA in the red nucleus of the rat without affecting the excitatory effects of quisqualate. CGS 19755 blocked the harmaline-induced increase in cerebellar cyclic GMP levels at a dose of 4 mg/kg i.p. with a duration of action exceeding 2 hr. CGS 19755 inhibited convulsions elicited by maximal electroshock in rat (ED50 = 3.8 mg/kg i.p. 1 hr after administration) and in mouse (ED50 = 2.0 mg/kg i.p. 0.5 hr after administration). Likewise, convulsions elicited by picrotoxin were inhibited by CGS 19755, whereas the compound was relatively weak in protecting against convulsions elicited by pentylenetetrazole or strychnine. CGS 19755 produced retention performance deficits in a dark avoidance task. However, CGS 19755 did not show a unique propensity for learning and memory disruption compared to other anticonvulsants.

Regulation of central cholecystokinin recognition sites by guanyl nucleotides.

Guanyl nucleotides affected the binding of radiolabeled cholecystokinin (CCK) octapeptide to rodent cortical binding sites. Micromolar quantities of a stable GTP analogue, guanylyl-imidodiphosphate (GppNp), resulted in a plateau where binding was decreased by 30%. In the presence of 25 microM GppNp, binding analysis revealed a decrease in affinity (increase in KD), without an apparent effect on the maximal number of binding sites. Ki values for CCK-related peptides shifted up to 1.6-fold. The rate of peptide association decreased by threefold, and the rapid component of peptide dissociation increased. The collective data suggest that a class of central CCK binding sites is linked to nucleotide regulatory proteins. The evidence is discussed with regard to multiple receptor populations and to possible interconversions between receptor types.

Inhibition of climbing and mossy fiber input to mouse cerebellar Purkinje cells by cholecystokinin.

Cholecystokinin (CCK)-8S was found both to decrease basal and to antagonize harmaline-dependent increases in cerebellar cyclic GMP (cGMP) in the mouse. These actions were not blocked by parenteral pretreatment with naloxone, proglumide or CR-1409, indicating a central-type CCK receptor activation with no involvement of opioid systems. These data were further confirmed by the greater potency (200x) of intraventricular relative to s.c. doses for CCK-8S. The intraventricular administration of CCK-4, t.BOC.CCK-4 and CCK-8US also resulted in decreased cerebellar cGMP levels, consistent with a central-type CCK receptor action. Direct administration of CCK-8S into the cerebellum failed to alter either basal or harmaline-stimulated cerebellar cGMP levels, indicating that the actions of this peptide on cerebellar cGMP are not directly at the level of the cerebellum. The convulsants, picrotoxin and pentylenetetrazol, elevated cerebellar cGMP with no antagonism by pretreatment with s.c. CCK-8S. In marked contrast, the increases in cerebellar cGMP induced by treatment with amphetamine, apomorphine, DN 1417, oxotremorine and harmaline were all antagonized by pretreatment with s.c. CCK-8S. These data are consistent with CCK receptor involvement in the regulation of climbing and mossy fiber input to the cerebellum. These actions apparently involve the central-type CCK receptor that resides outside of the cerebellum proper. The exact site(s) of action for CCK-8S remain(s) to be defined.

CPP, a selective N-methyl-D-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in vivo.

3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) was synthesized as a rigid analog of 2-amino-7-phosphonoheptanoate, a previously known antagonist at the N-methyl-D-aspartate (NMDA) preferring, or NMDA-type, of excitatory amino acid receptor. CPP was found to be a potent, selective and competitive antagonist of NMDA-type receptors. CPP antagonized with an IC50 of 8 muM [3H]ACh release which was evoked from rat striatal brain slices by NMDA (50 muM). In contrast, the release of [3H]ACh evoked by elevated KCI was not inhibited by CPP even at a concentration of 100 muM. The antagonism by CPP of NMDA-evoked [3H]ACh release was competitive, with a pA2 of 5.66 for CPP, compared with a pA2 value of 5.22 for 2-amino-7-phosphonoheptanoate. CPP affected neither the uptake of L-[3H]glutamate nor the inhibition by aconitine of L-[3H]glutamate uptake, suggesting a lack of membrane-stabilizing or local anesthetic effects, and also suggesting that CPP itself may not be taken up through the L-glutamate membrane transporter. Moreover, [3H] CPP was not accumulated by synaptosomes (P2 fraction) which avidly accumulate L-[3H]glutamate, supporting the concept that this NMDA-type receptor antagonist acts at an NMDA-type receptor on the external surface of the plasma membrane. CPP (10 muM) failed to interact with any of 21 other putative neurotransmitter receptors including alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding (quisqualate-type receptor) and [3H]kainate binding (kainate-type receptor). Audiogenic convulsions in DBA/2 mice were blocked by CPP (ED50 = 1.5 mg/kg i.p.) as were NMDA-induced seizures in CF-1 mice (ED50 = 1.9 mg/kg i.p.). In both strains, CPP impaired the traction reflex at higher doses (ED50 = 6.8 mg/kg and 6.1 mg/kg and 6.1 mg/kg i.p. for DBA/2 and CF-1, respectively). The traction reflex impairment by CPP may be due to muscle relaxant effects of the compound, an explanation supported by the finding that CPP reduced muscle tone as assessed by electromyogram measurement in animals whose muscle tone had been increased by opiate administration. Finally, cerebellar cyclic GMP levels, known to be sensitive to neurotransmission via NMDA-type receptors, were decreased by CPP (ED50 = 4.7 mg/kg i.p.) in mice. In conclusion, based upon the competitive antagonism by CPP of NMDA-evoked [3H] ACh release in vitro and the antagonism of NMDA-induced convulsions in vivo, the data presented are consistent with competitive antagonism of NMDA-type receptors.

Benzodiazepine interactions with central thyroid-releasing hormone binding sites: characterization and physiological significance.

Thyrotropin-releasing hormone (TRH) and several TRH analogs were examined in the [3H]-3-Me-His2-TRH ([3H]MeTRH) receptor-binding assay in rat amygdala, striatal and cortical membranes. The benzodiazepine, chlordiazepoxide, as reported in the literature was found to displace [3H]MeTRH with an IC50 value of 3.6 X 10(-7) M in amygdala membranes. Midazolam was, however, identified as being 6-fold more active than chlordiazepoxide with an IC50 value of 6.3 X 10(-8) M. The effect of these benzodiazepines on [3H]MeTRH binding did not appear to be related to their anxiolytic activity because the novel pyrazoloquinoline nonsedating anxiolytic, CGS 9896 was without effect on [3H]MeTRH binding at concentrations up to 1 X 10(-5) M. Chlordiazepoxide had similar activity in cortical membranes whereas midazolam was some 5 times less active in this preparation than in amygdala. Both compounds were weak displacers of [3H]MeTRH binding in striatal membranes, being at least two orders of magnitude less potent than in amygdala. In contrast TRH and its analogs, RX 77368 and DN-1417, were approximately 2 to 8 times more active in striatum than amygdala membranes. TRH and DN-1417 were less active in cortical membranes whereas RX 77368 was some three times more active than in striatum and amygdala. In three test procedures indicative of TRH agonist activity; thyroid-stimulating hormone release, reversal of pentobarbital sleeping time in mice and elevation of cerebellar cyclic GMP levels, the benzodiazepines were found to be devoid of activity, whereas TRH and related compounds produced their expected responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of central cholecystokinin receptors using a radioiodinated octapeptide probe.

We have developed a binding assay for 125I-Bolton-Hunter-labeled cholecystokinin octapeptide (125I-(BH)CCK8) using mouse cerebral cortex membrane preparations. This ligand interacts with cortical membrane preparations in a saturable, high-affinity manner, satisfying the requirements for specific cholecystokinin receptor labeling. Salt is required for maximal binding and BSA is specifically inhibitory with cerebral cortical but not with pancreatic sites. Cholecystokinin peptides as small as CCK30-33 displace binding at low nanomolar concentrations. Dissociation of 125I-(BH)CCK8 is biphasic in both mouse and guinea pig cortex. Pretreatment of membranes at 37 degrees C results in a marked loss of recognition sites, suggesting that the sites may be rapidly metabolized in vivo. After 37 degrees C pretreatment, the loss of CCK recognition sites corresponds to a selective loss of the slow component of dissociation curves. This selective elimination of one dissociation population, as well as the biphasic dissociation kinetics, suggests that at least two distinct CCK receptor subtypes exist in the brain.