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Glucosidic pathways of glycogen breakdown and glucose production by muscle from postexercised frogs.

Muscle and body glucose in frogs increases markedly during the initial hour of recovery after strenuous exercise. The liver is not the major source responsible for this accumulation. This is indicated by the stability of liver glycogen levels after exercise and by the observation that hepatectomized and normal frogs accumulate similar amounts of glucose in their muscles and body during recovery. The renal contribution cannot account for this increase in body glucose. Most of the glucose that accumulates in the body after exercise has a muscular origin, as indicated by the facts that two-thirds of the body glucose is found in muscle and that the intracellular levels of muscle glucose are much higher than those of the plasma. The glucose that accumulates outside muscle may also have a muscular origin. The glucosidic pathways of glycogen breakdown are the only metabolic avenue with sufficient capacity to account for the amount of glucose accumulated in muscle during the first hour of recovery. These results indicate that the ability of an isolated preparation of frog muscle to liberate glucose during recovery from exercise (Fournier et al. J. Biol. Chem. 267: 8234-8238, 1992) is not an artifactual metabolic curiosity but rather a metabolic reality that takes place in vivo. Glucose accumulation during recovery is thought to facilitate the metabolic transition of frog carbohydrate metabolism from a catabolic state, characteristic of exercise, to an anabolic one.

Analysis of Variance↗

Different modulation of steroidogenesis and prostaglandin production in frog ovary in vitro by ACE and ANG II.

Our aim was to study the role of angiotensin-converting enzyme (ACE) and angiotensin II (ANG II) on ovarian steroidogenesis and prostaglandin production of amphibian. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog angiotensin I (ANG I), and [Val5]ANG II were compared on frog ovaries of postreproductive and prereproductive periods. Very high ACE activity was found in ovary of water frog (Rana esculenta) compared with other frog tissues, and this activity was inhibited by the typical ACE inhibitors, captopril and lisinopril. Frog ovary tissue in postreproductive and prereproductive periods was incubated in vitro in the presence of ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val5]ANG II (1 microM), and synthetic bullfrog ANG I (1 microM). Production of 17 beta-estradiol, progesterone, androgens, and prostaglandins E2 and F2 alpha was determined. The data showed a modulation of 17 beta-estradiol, progesterone, and prostaglandin E2 production by ovary ACE; on the other hand, [Val5]ANG II modulated the production of progesterone and prostaglandin F2 alpha, whereas androgen production was not influenced. The present in vitro studies suggest the existence of two pathways independently regulated by ACE and ANG II modulating ovarian steroidogenesis and prostaglandin production.

Androgens↗

Sites and ionic mechanisms of hypoxic vasoconstriction in frog skin.

We tested the hypothesis that the cellular mechanisms mediating hypoxic vasoconstriction (HVC) in frog skin, an important vertebrate respiratory organ, are similar to those mediating HVC in the pulmonary vasculature of mammals. An accepted hypothesis in the lung is that alveolar hypoxia alters the redox potential in vascular smooth muscle cells of arterial vessels. This decreases membrane K+ conductance, causing depolarization. Depolarization increases the open probability of L-type Ca2+ channels, facilitating Ca2+ entry into the cell, which leads to vascular smooth muscle contraction and vasoconstriction. We studied the cutaneous microcirculation of the frog (Xenopus laevis) web by enclosing the web in a transparent chamber that was ventilated with different gas mixtures. Arteriolar and venular diameters were measured by video microscopy. Drugs were applied topically or intravascularly. A dose-dependent constriction to hypoxia occurred in arterioles but not venules, although both vessel types constricted to similar degrees to the thromboxane mimetic U-46619. The magnitude of HVC was not associated with arteriolar size. Constriction of arterioles with 4-amino pyridine, a K+-channel antagonist, was blocked by the L-type Ca2+-channel blocker nifedipine. Nifedipine also antagonized HVC and hypercapnic vasoconstriction. Bay K 8664, a drug that increases the open probability of L-type Ca2+ channels, augmented HVC. These data support our hypothesis that the cellular mechanisms mediating HVC are similar in frog skin and mammalian lungs. This similarity between amphibian and mammalian tissues suggests that the mechanisms of HVC may have arisen relatively early in vertebrate evolution. In addition, because of its structural simplicity and easy accessibility, frog skin may be a useful tissue for studying this general phenomenon in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Development of locomotor mechanisms in the frog.

Tadpoles swim by undulations of the body and tail, whereas frogs locomote by alternate (stepping) and synchronous (frog-kick) movements of the hindlimbs. The development of interlimb coordination was studied by recording the activity of hindlimb motoneurons from the left and right ninth ventral roots of the isolated central nervous system (CNS). Results showed that mechanisms responsible for interlimb coordination of stepping are functional when the hindlimb is still composed of undifferentiated mesenchyme and before the lateral motor column has stabilized (stage III). The early appearance of coordinated activation of hindlimb motoneurons suggests that innervation of appropriate target muscles is not a prerequisite for normal development of circuits that mediate interlimb coordination of stepping. Synchronous activation of left and right hindlimb motoneurons (fictive frog kicks) appeared later in development (stage XIV). Throughout larval development 1:1 frequency coupling between both alternating and synchronous bursts of hindlimb motoneurons and bursts of primary motoneurons (those innervating axial muscles) was found. Recordings of peripheral nerve activity showed that motoneurons innervating antagonistic muscles of the thigh burst in antiphase. This intralimb coordination was present at stage X, a foot paddle stage that was the earliest stage in which the peripheral nerves were successfully dissected. That the neural activity of the isolated nervous system described above indeed underlies coordinated locomotor movements of the hindlimbs was shown by single-frame videotape analysis of hindlimb movements produced by the otherwise isolated CNS. The stepping movements displayed by those preparations were consistent with patterns of electrophysiological burst activity recorded from the ventral roots and peripheral nerves. The ontogenetic sequence in which the different patterns of electrophysiological activity emerged is the same as that of the corresponding behaviors in the intact tadpole. Although there were developmental changes in the reliability with which coordinated activity in the ventral roots and peripheral nerves was observed, each mode of coordination remained qualitatively unchanged from its earliest appearance through metamorphosis. These results show that mechanisms underlying locomotor coordination of the hindlimbs develop very early in larval ontogeny of the frog and can function when isolated from the periphery.

Afferent Pathways↗

Neurotensin modulates the electrical activity of frog pituitary melanotropes via activation of a G-protein-coupled receptor pharmacologically related to both the NTS1 and nts2 receptors of mammals.

The primary structure of frog neurotensin (fNT) has recently been determined and it has been shown that fNT is a potent stimulator of alpha-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of fNT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of fNT. In the cell-attached configuration, fNT (10(-7) M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10(-7) M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10(-10) to 10(-6) M) of fNT or the C-terminal hexapeptide NT(8-13) caused a dose-dependent increase in the frequency of action potentials with EC(50) of 2 x 10(-8) and 5 x 10(-9) M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp(11)]NT(8-13) > Boc-[D-Trp(11)]NT(8-13) > Boc-[Lys(8,9), Nal(11)]NT(8-13) > Boc-[Psi11,12]NT(8-13). In contrast, the cyclic pseudopeptide analogs of NT(8-13), Lys-Lys-Pro-D-Trp-Ile-Leu and Lys-Lys-Pro-D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10(-5) M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10(-5) M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10(-6) M) had no effect on the basal electrical activity and the response of melanotropes to fNT. In cells which were dialyzed with guanosine-5'-O-(3-thiotriphosphate) (10(-4) M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5'-O-(2-thiodiphosphate) (10(-4) M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 microg/ml) or pertussis toxin (0.2 microg/ml) did not affect the electrical response to fNT. Intracellular application of the G(o/i/s) protein antagonist GPAnt-1 (3 x 10(-5) M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G(q/11) protein antagonist GPAnt-2A (3 x 10(-5) M) abrogated the response to fNT. The present data demonstrate that fNT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by fNT can be accounted for by activation of a G(q/11)-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.

Adamantane↗

The evolution of neural circuits controlling feeding behavior in frogs.

Our approach to understanding motor systems is a phylogenetic, 'outside-in' approach, the goal of which is to identify behavioral transitions during phylogenesis and elucidate their neurological basis. In this paper, we review the results of recent behavioral, biomechanical and neurological studies on frog feeding behavior. These studies show that highly protrusible tongues have evolved numerous times independently among frogs, and that the biomechanics and neuromuscular control of feeding behavior have been transformed repeatedly during frog evolution. Many of the independent lineages possess unique biomechanical mechanisms for protracting their tongues and unique neural mechanisms for coordinating feeding behavior. In frogs, there has been considerable evolution at the interface between reticular central pattern generators (CPGs) associated with feeding and sensory feedback circuits that modulate feeding motor output. In particular, the roles of hypoglossal and glossopharyngeal sensory feedback appear to have been relatively plastic in their evolution. Prey-type dependence of hypoglossal sensory feedback in Rana suggests that the interaction between descending visual control and sensory feedback also may be evolutionarily plastic. Comparative studies have found that motor systems sometimes evolve conservatively across morphological and behavioral transitions (i.e., the shoulder in birds) or, alternatively, they may be subject to considerably more evolutionary change than is reflected in morphological characteristics (i.e., feeding in cichlids). We hypothesize that the CPG circuits for feeding behavior in the reticular formation may evolve conservatively because they are highly integrated, multifunctional networks which cannot be optimized for one function without compromising others. In contrast, the interfaces between the CPG, sensory feedback and descending control should be less constrained. When changes in motor patterns occur during evolution, it is likely that sensory feedback or descending control may be involved.

Animals↗

Alteration of the hypothalamic-pituitary-gonadal axis in estrogen- and androgen-treated adult male leopard frog, Rana pipiens.

BACKGROUND: Gonadal steroids, in particular 5 alpha-dihydrotestosterone (DHT) and 17 beta-estradiol (E2), have been shown to feed back on the hypothalamic-pituitary-gonadal (HPG) axis of the ranid frog. However, questions still remain on how DHT and E2 impact two of the less-studied components of the ranid HPG axis, the hypothalamus and the gonad, and if the feedback effects are consistently negative. Thus, the goal of the study was to examine the effects of DHT and E2 upon the HPG axis of the gonadally-intact, sexually mature male leopard frogs, Rana pipiens. METHODS: R. pipiens were implanted with silastic capsules containing either cholesterol (Ch, a control), DHT, or E2 for 10 or 30 days. At each time point, steroid-induced changes in hypothalamic GnRH and pituitary LH concentrations, circulating luteinizing hormone (LH), and testicular histology were examined. RESULTS: Frogs implanted with DHT or E2 for 10 days did not show significant alterations in the HPG axis. In contrast, frogs implanted with hormones for 30 days had significantly lower circulating LH (for both DHT and E2), decreased pituitary LH concentration (for E2 only), and disrupted spermatogenesis (for both DHT and E2). The disruption of spermatogenesis was qualitatively similar between DHT and E2, although the effects of E2 were consistently more potent. In both DHT and E2-treated animals, a marked loss of all pre-meiotic germ cells was observed, although the loss of secondary spermatogonia appeared to be the primary cause of disrupted spermatogenesis. Unexpectedly, the presence of post-meiotic germ cells was either unaffected or enhanced by DHT or E2 treatment. CONCLUSIONS: Overall, these results showed that both DHT and E2 inhibited circulating LH and disrupted spermatogenesis progressively in a time-dependent manner, with the longer duration of treatment producing the more pronounced effects. Further, the feedback effects exerted by both steroid hormones upon the HPG axis were largely negative, although the possibility exists for a stimulatory effect upon the post-meiotic germ cells.

Animals↗

Immunocytochemical and phylogenetic distribution of aquaporins in the frog ventral skin and urinary bladder.

We recently cloned three cDNAs encoding frog aquaporin (AQP-h1, BAC07470; AQP-h2, BAC82379; and AQP-h3, BAC07471) from the ventral pelvic skin of the tree frog, Hyla japonica. The present study demonstrated that Hyla AQP-h2 was translocated from cytoplasmic pools to the apical plasma membranes of the granular cells in the bladder after antidiuretic hormone stimulation and that Hyla AQP-h2 and AQP-h3 behaved similarly in the ventral pelvic skin. Further, we found that terrestrial and tree frogs, but not aquatic and semiterrestrial-adapted frogs, absorbed water from their ventral pelvic skin by AQP-h3-like protein in concert with AQP-h2-like protein.

Animals↗

Expression and processing of the [Pro(2),Met(13)]somatostatin-14 precursor in the intermediate lobe of the frog pituitary.

The biosynthesis of various hypothalamic neuropeptides has been previously reported in anterior pituitary cells but not in intermediate lobe cells. We have recently demonstrated the occurrence of two somatostatin isoforms in the frog brain, namely somatostatin-14 (SS1) and [Pro(2),Met(13)]somatostatin-14 (SS2). In the present study, we demonstrate that the gene encoding the SS2 precursor (PSS2) is actively expressed in the intermediate lobe of the frog pituitary. High concentrations of PSS2 mRNA have been detected by Northern blot analysis and in situ hybridization in the frog pars intermedia but not in the pars distalis or pars nervosa. The distribution of PSS1- and PSS2-derived peptides has been investigated by immunohistochemistry using two antisera directed against SS1 and the sequence 54-66 of PSS2 (PSS2(54-66)), respectively. The SS1 antiserum stained only a network of fibers in the neural lobe and a few nerve processes in the intermediate lobe. In contrast, the PSS2(54-66) antiserum produced intense labeling of melanotrope cells in the pars intermedia. Biochemical characterization of the immunoreactive materials present in pituitary extracts was performed by combining high-performance liquid chromatography analysis and RIA detection. The SS1 RIA revealed the existence of two major immunoreactive peaks that exhibited the same retention times as synthetic SS1 and SS2. The PSS2(54-66) RIA detected a single peak that likely corresponds to the N-flanking peptide of SS2 (PSS2(1-66)). The present study reveals that melanotrope cells of the frog pituitary selectively express the PSS2 gene and fully process PSS2 to generate the mature somatostatin variant SS2. Taken together, these data provide the first evidence that the gene encoding a hypophysiotropic neuropeptide is intensely expressed in the intermediate lobe of the pituitary.

Animals↗

Neurotensin stimulates both calcium mobilization from inositol trisphosphate-sensitive intracellular stores and calcium influx through membrane channels in frog pituitary melanotrophs.

Neurotensin (NT) is a potent stimulator of electrical and secretory activities in frog pituitary melanotrophs. The aim of the present study was to characterize the transduction pathways associated with activation of NT receptors in frog melanotrophs. Application of synthetic frog NT (fNT) increased the cytosolic calcium concentration ([Ca2+]c) and stimulated the formation of inositol trisphosphate (IP3). The phospholipase C inhibitor U-73122 blocked the electrophysiological and secretory effects of fNT. Intracellular application of the IP3 receptor antagonist heparin abolished fNT-induced electrical activity. Suppression of Ca2+ in the incubation medium markedly reduced the effect of NT on [Ca2+]c, firing rate, and alpha-melanocyte-stimulating hormone (alphaMSH) secretion. Similarly, the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels, 2-Aminoethoxydiphenylborane, and the nonselective Ca2+ channel blockers GdCl3 and NiCl2, attenuated the [Ca2+]c increase and the electrical and secretory responses evoked by fNT. Coapplication of the L- and N-type Ca2+ channel blockers nifedipine and omega-CgTx GVIA reduced the effects of fNT on action potential discharge, [Ca2+]c increase, and alphaMSH release. The protein kinase C (PKC) inhibitors, PKC-(19-31) and chelerythrine, reduced the electrophysiological and secretory responses induced by iterative applications of fNT. Collectively, these results demonstrate that, in frog melanotrophs, NT stimulates the phospholipase C/PKC pathway and increases [Ca2+]c. Both Ca2+ release from intracellular stores and Ca2+ influx through L- and N-type Ca2+ channels are involved in fNT-induced alphaMSH secretion. In addition, the present data indicate that PKC plays a crucial role in maintenance of the responsiveness of melanotrophs to NT.

Animals↗

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Amino Acid Sequence↗

Acetylcholine stimulates alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs through activation of muscarinic and nicotinic receptors.

The release of alpha MSH from the pars intermedia of amphibians is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In this study we have examined the possible involvement of acetylcholine (ACh) in the regulation of alpha MSH secretion from the pars intermedia of the frog (Rana ridibunda) using the perifusion technique. When intact neurointermediate lobes (NIL) were exposed to graded doses of ACh (3 X 10(-7) to 3 X 10(-4) M), a dose-dependent stimulation of alpha MSH release was observed. Repeated administration of ACh (10(-4) M) induced reproducible responses of NIL without any desensitization phenomenon. ACh was also capable of stimulating alpha MSH release from dispersed intermediate lobe cells, indicating that the neurotransmitter exerts its effect by acting directly on frog melanotrophs. Using the monoclonal antibody M-35 against calf muscarinic receptors we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in the frog pars intermedia. The stimulatory action of ACh was mimicked by both nicotine and muscarine (10(-5) M each). Nicotine-induced stimulation of alpha MSH release was partially abolished by alpha-bungarotoxin (10(-6) M) and hexamethonium (10(-4) M). The stimulatory effect of muscarine was suppressed by atropine and the M1-muscarinic antagonist pirenzepine (10(-5) M), but not by the M2-muscarinic antagonist gallamine. We have investigated the effect of ACh during administration of specific nicotinic and muscarinic antagonists. While hexomethonium or atropine could block only part of the stimulatory effect of ACh, concomitant administration of these antagonists totally abolished the response of NIL to ACh. Finally, the stimulatory effect of ACh was not impaired during prolonged administration of the beta-adrenergic antagonist propranolol. These data show that ACh stimulates in vitro alpha MSH secretion by frog NIL. Our results also indicate that amphibian pars intermedia cells possess two types of cholinergic receptors, an M1-muscarinic receptor sensitive to pirenzepine and nicotinic receptors sensitive to hexamethonium and alpha-bungarotoxin.

Acetylcholine↗

Pharmacological characterization of vasotocin stimulation of phosphoinositide turnover in frog adrenal gland.

In a previous report we demonstrated the presence of a vasotocin (AVT)-like peptide in chromaffin cells of the amphibian adrenal gland and showed that synthetic AVT is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells. In the present study we evaluated the relative potency of various AVT analogs and investigated the mechanism of action of AVT on frog interrenal (adrenal) tissue. Several AVT agonists, including hydrin 2, oxytocin (OXT), arginine vasopressin (AVP), Lys-conopressin G, and mesotocin (MT), were able to mimic the stimulatory effect of AVT on steroid secretion, but AVT was by far the most potent stimulator of steroidogenesis. In the series of analogs studied, the order of potency was: AVT greater than hydrin 2 greater than OXT greater than AVP greater than Lys-conopressin G greater than MT greater than [deamino-Cys1,D-Arg8]AVP greater than [d(CH2)5,Tyr(OMe)2] AVP. The effect of AVT (5 x 10(-10) M) was totally blocked by both the antidiuretic V2 antagonist [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP (10(-6) M) and the oxytocinergic antagonist [d(CH2)5,Tyr(OMe)2,Orn8]AVT (10(-6) M); the V2 antagonist was approximately twice as potent as the OXT antagonist. In contrast, the V1 antagonist 1-(1-mercapto-4-phenylcyclohexaneacetic acid)-AVP (10(-6) M) did not affect the response of the interrenal tissue to AVT. Indomethacin (5 microM), a cyclooxygenase inhibitor, induced a dramatic decrease in the spontaneous secretion of corticosteroids, but did not impair the stimulatory effect of AVT (5 x 10(-9) M) on corticosterone and aldosterone secretion. In addition, AVT did not stimulate the production of prostaglandin E2, suggesting that prostaglandins are not involved in the mechanism of action of AVT. Concurrently, AVT did not modify cAMP production by frog adrenal slices. In contrast, AVT induced both an increase in inositolphosphate production and a reduction of membrane phospholipid content. We conclude that in the frog adrenal gland, the stimulatory effect of AVT on steroid secretion is mediated through activation of receptors related to the mammalian V2 and/or OXT receptors, which are positively coupled to phosphoinositide-specific phospholipase C.

Adrenal Cortex Hormones↗

Isolation, primary structure, and effects on alpha-melanocyte-stimulating hormone release of frog neurotensin.

Neurotensin (NT) was isolated in pure form from the small intestine of the European green frog, Rana ridibunda, and its primary structure was established as pGlu-Ala-His-Ile-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This sequence contains five amino acid substitutions (Leu2-->Ala, Tyr3-->His, Glu4-->Ile, Asn5-->Ser, and Pro7-->Ala) compared with human NT. A peptide with identical chromatographic properties was identified in an extract of frog brain. Synthetic frog NT produced a concentration-dependent increase in alphaMSH release from perifused frog pars intermedia cells, with an ED50 of 5 x 10(-9) M. A maximum response (276.3 +/- 45.5% above basal release) was produced by a 10(-8) M concentration. Repeated administration of NT to melanotrope cells revealed the occurrence of a rapid and pronounced desensitization mechanism. The data are consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.

Amino Acid Sequence↗

Frog chromogranin A messenger ribonucleic acid encodes three highly conserved peptides. Coordinate regulation of proopiomelanocortin and chromogranin A gene expression in the pars intermedia of the pituitary during background color adaptation.

Chromogranin A (CgA) is a neuroendocrine secretory protein that is widely used as a marker for endocrine neoplasms but whose function is not completely understood. In mammals, it is thought that CgA is a precursor for biologically active peptides. Here, we describe the cloning of a complementary DNA encoding CgA from a nonmammalian vertebrate, the frog Rana ridibunda. Sequence analysis revealed that frog CgA exhibits only 40-44% amino acid sequence similarity with its mammalian homologues. The amino acid identity is confined to three regions (70-80% identity) of the protein that are flanked by conserved pairs of basic amino acid residues, suggesting that proteolytic processing at these cleavage sites may give rise to three biologically active peptides whose sequences have been highly preserved during evolution. Tissue distribution analysis by Northern blot and in situ hybridization revealed the widespread expression of frog CgA messenger RNA in the brain and in endocrine tissues, the highest concentration occurring in the distal lobe of the pituitary. Adaptation of frog skin color to a dark background caused a concomitant increase in CgA and POMC messenger RNA levels in the intermediate lobe of the pituitary. Taken together, these data indicate that CgA may function as a precursor to three highly conserved peptides that may exert regulatory functions in the neuroendocrine system.

Adaptation, Physiological↗

Molecular cloning of growth hormone-releasing hormone/pituitary adenylyl cyclase-activating polypeptide in the frog Xenopus laevis: brain distribution and regulation after castration.

Pituitary adenylyl cyclase-activating peptide (PACAP) appears to regulate several neuroendocrine functions in the frog, but its messenger RNA (mRNA) structure and brain distribution are unknown. To understand the potential role of PACAP in the male frog hypothalamic-pituitary-gonadal axis, we cloned the frog Xenopus laevis PACAP mRNA and determined its distribution in the brain. We then analyzed the castration-induced alterations of mRNA expression for PACAP and its selective type I receptor (PAC1) in the hypothalamic anterior preoptic area, a region known to regulate reproductive function. The PACAP mRNA encodes a peptide precursor predicted to give rise to both GH-releasing hormone and PACAP. The deduced peptide sequence of PACAP-38 was nearly identical to that of human PACAP with one amino acid substitution. Abundant PACAP mRNA was detected in the brain, but not several other tissues, including the testis. In situ hybridization revealed strong expression of the PACAP gene in the dorsal pallium, ventral hypothalamus, and nuclei of cerebellum. PACAP mRNA signals were weak to moderate in the hypothalamic anterior preoptic area and were absent in the pituitary. Castration induced an increase in the expression of PACAP and PAC1 receptor mRNAs in the hypothalamic anterior preoptic area after 3 days. Replacement with testosterone prevented the castration-induced changes. These results provide a molecular basis for studying the physiological functions of PACAP in frog brain and suggest that PACAP may be involved in the feedback regulation of hypothalamic-pituitary-gonadal axis.

Amino Acid Sequence↗

Mechanism of tongue protraction in microhylid frogs.

High-speed videography and muscle denervation experiments were used to elucidate the mechanism of tongue protraction in the microhylid frog Phrynomantis bifasciatus. Unlike most frogs, Phrynomantis has the ability to protract the tongue through a lateral arc of over 200 degrees in the frontal plane. Thus, the tongue can be aimed side to side, independently of head and jaw movements. Denervation experiments demonstrate that the m. genioglossus complex controls lateral tongue aiming with a hydrostatic mechanism. After unilateral denervation of the m. genioglossus complex, the tongue can only be protracted towards the denervated (inactive) side and the range through which the tongue can be aimed is reduced by 75%. Histological sections of the tongue reveal a compartment of perpendicularly arranged muscle fibers, the m. genioglossus dorsoventralis. This compartment, in conjunction with the surrounding connective tissue, generates hydrostatic pressure that powers tongue movements in Phrynomantis. A survey of aiming abilities in 17 additional species of microhylid frogs, representing a total of 12 genera and six subfamilies, indicates that hydrostatic tongues are found throughout this family. Among frogs, this mechanism of tongue protraction was previously known only in Hemisus and may represent a synapomorphy of Hemisus and Microhylidae.

Animals↗

Inhibition of the increased 17beta-estradiol-induced mast cell number by melatonin in the testis of the frog Rana esculenta, in vivo and in vitro.

In the present study, we have utilized 17beta-estradiol to induce the increase of mast cell number in order to verify the melatonin effect on mast cell accumulation in the frog testicular interstitium. Data obtained from in vivo experiments confirm that 17beta-estradiol increases the mast cell number and indicate a melatonin-inhibitory role in their accumulation in the frog testis. In addition, melatonin interferes with the effects of estradiol on the increase of mast cell number in short-term cultured testes, and this result has also been obtained in a dose-response experiment at physiological concentration. The data suggest that melatonin acts on mast cell number directly via its local action in the frog gonads. In conclusion, our study shows, for the first time, that melatonin may interfere, probably via estrogen receptors, with the differentiation and/or proliferation of mast cells induced by estradiol treatment either in vivo or in vitro in the testis of the frog Rana esculenta.

Animals↗