Evolution of gnathostome prodynorphin and proenkephalin: characterization of a shark proenkephalin and prodynorphin cDNAs

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark α-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkephalin precursor sequences. Shark prodynorphin encodes both kappa (α-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins).     

Conversion of thyroxine to triiodothyronine in an adult amphibian, Bufo marinus

Adult toads (Bufo marinus) were injected with T₄ (50 μg/day for 5 days) to assess possible conversion of T₄ to T₃. Plasma T₃ was undetectable (<12.5 ng/dl) before injection of T₄; the mean corrected plasma level of T₃ after injection was 259 ± 83 (SD) ng/dl, indicating in vivo conversion of T₄ to T₃ in an adult amphibian. Although as yet of uncertain physiologic significance, this conversion may be important in the action of thyroid hormone in adult amphibia.     

Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain.

Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.     

Cloning of prodynorphin cDNAs from the brain of Australian and African lungfish: implications for the evolution of the prodynorphin gene

In mammals the opioids Met-enkephalin and Leu-enkephalin are derived from a common precursor, proenkephalin, and as a result these neuropeptides are co-localized in enkephalinergic neurons. The mammalian scheme for enkephalinergic networks is not universal for all classes of sarcopterygian vertebrates. In an earlier study, distinct Met- and Leu-enkephalin-positive neurons were detected in the central nervous system (CNS) of the African lungfish, Protopterus annectens. More recently, characterization of proenkephalin cDNAs separately cloned from the CNS of P. annectens and the Australian lungfish, Neoceratodus forsteri, revealed that the proenkephalin gene in these species encodes only Met-enkephalin-related opioids. In the current study a full-length prodynorphin cDNA (accession No. AY 445637) was cloned and sequenced from the CNS of N. forsteri. In addition to encoding alpha-neoendorphin, dynorphin A and dynorphin B sequences unique to the lungfish, two Leu-enkephalin sequences, flanked by paired basic amino acid proteolytic cleavage sites, were detected in this precursor. The partial sequence of a P. annectens prodynorphin cDNA (accession No. AY445638) also encoded a Leu-enkephalin sequence and a novel YGGFF sequence. The presence of the Leu-enkephalin sequence in the lungfish prodynorphin precursors would explain the origin of the distinct Leu-enkephalin-positive neurons found in the African lungfish CNS. The realization that Met-enkephalin and Leu-enkephalin can be derived from distinct opioid-coding precursor genes calls into question the interpretation of comparative immunohistochemical studies that have mapped 'enkephalinergic' networks in non-mammalian vertebrates.     

Evidence for a differential postnatal development of proenkephalin B (= prodynorphin)-derived opioid peptides in the rat hypothalamus

[he concentrations of immunoreactive (ir-) peptides derived from the opioid peptide precursors proenkephalin A (Met-enkephalin), proenkephalin B [dynorphin (DYN)-(1-17), dynorphin-(1-8), dynorphin B, alpha-neoendorphin (alpha-NEO-E), beta-NEO-E] and proopiomelanocortin [beta-endorphin (beta-END)], and of the neurosecretory hormones vasopressin and oxytocin increased between approximately 10-fold and 50-fold from birth to adulthood in the rate hypothalamus. Gel filtration and HPLC analysis of proenkephalin B-derived opioid peptides revealed that in 3-day-old rats the predominant portion of ir-dynorphin-(1-17) and a substantial part of ir-dynorphin B consisted of a high (6000) mol wt species, a common precursor peptide for DYN-(1-17) and DYN B. In adults rats, however, authentic DYN-(1-17) and DYN B were found to be the major ir-forms. The mol wt patterns of ir-DYN-(1-8), ir-alpha-NEO-E and ir-beta-NEO-E did not differ between 3-day-old and adult rats and reflected predominantly the respective authentic opioid peptides. Taking into consideration the developmental changes in the mol wt pattern of ir-DYN-(1-17), authentic DYN-(1-17) was 5 times lower in concentration than DYN-(1-8) in 3-day-old rats, whereas in adults these opioid peptides occurred in equimolar concentrations. These findings suggest that the posttranslational processing of the precursor proenkephalin B changes in the course of postnatal development. Ir-beta-END in the hypothalamus of newborn and adult rats consisted exclusively of beta-END-sized peptides which were not (unlike those in the intermediate pituitary lobe) alpha-N-acetylated. Thus, in the hypothalamus, the enzymatic processing of the opioid peptide precursor proopiomelanocortin to beta-END seems to be fully active at birth, in contrast to that of proenkephalin B.     

Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase.

The complete amino acid sequence for the type I isozyme of hexokinase from rat brain has been deduced from the nucleotide sequence of cloned cDNA. The nucleotide sequence of 91 bases in the 5' untranslated region as well as that of the entire 3' untranslated region preceding the poly(A) sequence have also been determined. The N- and C-terminal halves of brain hexokinase show extensive sequence similarity to each other and to yeast hexokinase. These results provide direct support for the proposal that the mammalian hexokinases of approximately 100 kDa have evolved by a process of duplication and fusion of a gene encoding an ancestral hexokinase similar to the yeast enzyme of approximately 50 kDa. Taking this similarity in sequence to indicate basic similarity in structure between the N- and C-terminal regions of brain hexokinase and the yeast enzyme, a proposed structure for the mammalian hexokinase has been developed by fusing two molecules of yeast hexokinase, whose structure has previously been determined by x-ray crystallographic studies. Various features of the model are shown to be consistent with experimental observations bearing on the structure of the brain enzyme.     

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark,Heterodontus portusjacksoni

Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.     

Cloning proenkephalin from the brain of a urodele amphibian (Taricha granulosa) using a DOR-specific primer in a 3'RACE reaction

A large cDNA fragment that codes for proenkephalin (PENK) was cloned from the rough-skinned newt, Taricha granulosa (GenBank Accession: AY817670). This 1299-bp PENK cDNA extends from the poly(A) sequence on the 3′ end into the 5′-UTR (221 bp) upstream of an open reading frame that codes for 264 amino acids and a stop codon. Within the precursor are five Met-enkephalin sequences and two C-terminally extended forms of Met-enkephalin (YGGFMRGV and YGGFMRY). The organization of the opioid core sequences within the newt PENK closely resembles that reported for other vertebrates. In this urodele amphibian, as in anurans, PENK does not contain the penultimate Leu-enkephalin opioid sequence found in mammals, and instead has in this position Met-enkephalin. PENK cDNA was amplified from newt brain in a RACE PCR targeting the 3′ end of the newt delta opioid receptor (DOR). It remains to be determined whether generating the cDNA for the newt PENK while cloning its receptor was serendipitous or the result of a meaningful coincidence between the DOR and PENK sequences.     

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.     

Delta and mu opioid receptors from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa: cloning, heterologous expression, and pharmacological characterization

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.     

Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning, and expression in the brain.

Three peptides derived from diazepam-binding inhibitor (DBI) were isolated in pure form from the brain of the frog Rana ridibunda. The primary structures of these peptides showed that they correspond to mammalian DBI-(1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate primers, whose design was based on the amino acid sequence data, was used to screen a frog brain cDNA library. The cloned cDNA encodes an 87-amino acid polypeptide, which exhibits 68% similarity with porcine and bovine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at positions 14-15 and 62-63 and a single cysteine within the biologically active region of the molecule. Northern blot analysis showed that DBI mRNA is expressed at a high level in the brain but is virtually absent in peripheral tissues. The distribution of DBI mRNA and DBI-like immunoreactivity in the frog brain was studied by in situ hybridization and immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the ventricular cavity. Since amphibia diverged from mammals at least 250 million years ago, the data show that evolutionary pressure has acted to conserve the structure of DBI in the vertebrate phylum. The distribution of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.     

In the african lungfish Met-enkephalin and leu-enkephalin are derived from separate genes: cloning of a proenkephalin cDNA

A full-length proenkephalin cDNA (accession number: AF232670) was cloned from an African lungfish (Protopterus annectens) brain cDNA library. The 1,351-bp African lungfish proenkephalin contains an open reading frame that codes 266 amino acids and a stop codon. Within the sequence of lungfish proenkephalin there are 5 pentapeptide opioid sequences (all YGGFM), 1 octapeptide opioid sequence (YGGFMRSL) and 1 heptapeptide opioid sequence (YGGFMGY). A Leu-enkephalin sequence was conspicuously absent in lungfish proenkephalin. These results, coupled with observations on the organization of amphibian proenkephalin and mammalian proenkephalin, indicate that among the Sarcopterygii (lobed finned fish and tetrapods), the appearance of a Leu-enkephalin sequence in proenkephalin may have evolved in either the ancestral amniotes or the ancestral mammals, but not earlier in sarcopterygian evolution. Furthermore, the detection of neurons in the lungfish CNS that are only immunopositive for Met-enkephalin, coupled with earlier anatomical studies on the presence of neurons in the lungfish CNS that are only immunopositive for Leu-enkephalin, indicates that a Leu-enkephalin-coding opioid gene must be present in the CNS of the lungfish. This gene may be the lungfish form of prodynorphin. Given the phylogenetic position of the lungfish in vertebrate evolution, the putative Leu-enkephalin-coding gene must have evolved in the ancestral sarcopterygian vertebrates, or in the ancestral gnathostomes. The apparent slow rate of lungfish evolution makes these organisms interesting models for investigating the evolution of the opioid/orphanin gene family.     

Identification of a cDNA encoding a novel amphibian growth hormone-releasing peptide and localization of its transcript

Recently, we identified in the bullfrog brain a novel neuropeptide with a C-terminal Leu-Pro-Leu-Arg-Phe-NH(2) sequence. This amphibian neuropeptide was shown to stimulate growth hormone (GH) release in vitro and in vivo and so was designated frog GH-releasing peptide (fGRP). In this study, we cloned a cDNA encoding fGRP from the bullfrog brain by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced fGRP precursor consisted of 221 amino acid residues, encoding one fGRP and three putative fGRP-related peptides that included Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Northern blot analysis detected a single band of approximately 1.0 kb, indicating that no alternatively spliced forms were present. Such an apparent migration was in agreement with the estimated length of the cDNA, 902 bp. In situ hybridization further revealed the cellular localization of fGRP mRNA in the suprachiasmatic nucleus in the hypothalamus. In addition to fGRP, its related peptides may be hypothalamic factors involved in pituitary hormone secretion.     

Effects of central and peripheral chemoreceptor stimulation on ventilation in the marine toad, Bufo marinus

The contributions of central and peripheral chemoreceptors to respiratory control in lightly anesthetized Bufo marinus, were assessed by measuring the ventilatory responses to unidirectional ventilation (UDV) of the lungs at several concentrations of CO2 or O2, during intracranial perfusion (ICP) with hypercapnic acidic (5% CO2, pH 7.2) or hypocapnic alkaline (0% CO2, pH 8.3) mock CSF solutions. Peripheral chemoreceptor stimulation alone (hypoxia or hypercapnia during ICP with hypocapnic alkaline CSF) significantly increased breathing frequency and amplitude. ICP with hypercapnic acidic CSF further stimulated ventilation, primarily by significantly increasing the number of breaths/bout of breathing and decreasing the non-ventilatory time at all levels of peripheral ventilatory drive. When peripheral and central chemoreceptor stimulation was low toads were apneic. Stimulation of either central or peripheral chemoreceptors was sufficient to reinitiate breathing. Responses to ICP were greatest when perfusion was directed to the ventral medullary surface (VMS). These results suggest that the initiation of breathing and overall levels of breathing are functions of the combined afferent input from peripheral chemoreceptors and central CO2/pH sensitive chemoreceptors, located near the VMS. Stimulation of central chemoreceptors, however, produced longer duration bouts of rhythmic breathing than did peripheral chemoreceptor stimulation.     

Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin.

Two enkephalin-containing polypeptides of 3600 and 4900 daltons have been isolated from extracts of bovine adrenal medulla, purified to homogeneity, and analyzed by a combination of automated Edman degradation and enzymatic time course hydrolysis. The 4900-dalton polypeptide contains two copies of enkephalin, one an internal [Met]enkephalin sequence, the other a [Leu]enkephalin sequence at the carboxyl terminus. Sequence analysis of the 3600-dalton polypeptide has not been completed, but the polypeptide has been shown to contain a single [Met]enkephalin sequence followed by an -Arg-Phe linkage that forms the carboxyl terminus of the molecule. On the basis of these and other findings, we propose that the above enkephalin-containing polypeptides are intermediates in the biosynthesis of the enkephalins and that they are generated by posttranslational processing from a large multivalent enkephalin precursor molecule, proenkephalin. The term "multivalent" is used to indicate a polypeptide with many identical functional sequences.     

Distribution and characterization of natriuretic peptide receptors in the kidney of the toad, Bufo marinus.

The location and characteristics of atrial natriuretic peptide binding sites in the kidney of the toad, Bufo marinus, were determined. Specific (125)I-rANP binding sites were observed on glomeruli and blood vessels, but little if any binding was observed over regions corresponding to the renal tubules. (125)I-rANP binding in tissue sections and/or isolated membranes was completely displaced in the presence of 1 microM rat ANP, frog ANP, and porcine C-type natriuretic peptide (membranes only); however, residual binding remained after incubation with 1 microM of the NPR-C ligand, C-ANF, indicating the presence of two distinct binding sites. Electrophoresis of kidney membranes cross-linked to (125)I-rANP identified specific bands at approximately 70 and 140 kDa which correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP production rates in membrane preparations, while C-ANF had no stimulatory effect. Two partial cDNA clones generated using primers based on conserved regions of vertebrate natriuretic peptide receptors showed high homology to an NPR-C and the natriuretic peptide guanylate cyclase receptors (NPR-GC), respectively. This study provides evidence that the kidney of B. marinus contains both NPR-C and NPR-GC and that the glomerulus is potentially the principal site of ANP regulation in the kidneys.     

Alpha2 adrenergic receptors and the central control of breathing in the cane toad, Bufo marinus

The effect of adrenergic agents on the central control of breathing in the cane toad, Bufo marinus, was tested by applying adrenergic agents to the ventral medullary surface of decerebrate adult toads. Toads were unidirectionally ventilated while recording lung, buccal, and artery blood pressures (BPI), as well as heart rate (HR). Following a control period, filter paper pledgets soaked in the appropriate solution (epinephrine - 0.023, 0.05, 0.10, and 0.23 mM; norepinephrine - 0.002, 0.016, 0.032, and 0.16 mM; clonidine - 0.00375, 0.0375, and 0.375 mM; or yohimbine - 0.43) mM) were placed bilaterally on the ventral medullary surface. Epinephrine significantly increased the number of breaths (26%), lung amplitude (9%), and episode duration (21%), but had no effect on BP or HR. The alpha2-agonist. clonidine, significantly increased respiratory activity at moderate doses (0.0375 mM) and decreased activity at high doses (0.375 mM), however, it failed to elicit significant changes in BP or HR. Pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (0.43 mM), blocked the clonidine induced changes in respiratory activity. Yohimbine had no effect on cardiorespiratory parameters. Norepinephrine had no effect on either cardiovascular or respiratory variables. Thus, it appears that an alpha2 adrenergic mechanism is involved in the central control of respiration in this lower vertebrate.