Activation of a chondrocyte volume-sensitive Cl(-) conductance prior to macroscopic cartilage lesion formation in the rabbit knee anterior cruciate ligament transection osteoarthritis model.

OBJECTIVE: The anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl(-) current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model. DESIGN: Adult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol. RESULTS: At 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na(+), K(+) and Ca(2+) currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance. CONCLUSION: Changes in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.

Sodefrin: a female-attracting peptide pheromone in newt cloacal glands.

A decapeptide called sodefrin was isolated from the abdominal gland of the cloaca of the male red-bellied newt, Cynops pyrrhogaster. The native peptide, as well as the synthetic one, had a female-attracting activity. Sodefrin was found in the apical portion of the epithelial cells of the abdominal gland. Sodefrin is apparently species specific because it did not attract females of Cynops ensicauda. This is the first amphibian pheromone to be identified and the first peptide pheromone identified in a vertebrate.

Stimulatory action of protein kinase C(epsilon) isoform on the slow component of delayed rectifier K+ current in guinea-pig atrial myocytes.

BACKGROUND AND PURPOSE: Protein kinase C (PKC) comprises at least twelve isoforms and has an isoform-specific action on cardiac electrical activity. The slow component of delayed rectifier K(+) current (I (Ks)) is one of the major repolarizing currents in the hearts of many species and is also potentiated by PKC activation. Little is known, however, about PKC isoform(s) functionally involved in the potentiation of I (Ks) in native cardiac myocytes. EXPERIMENTAL APPROACH: I (Ks) was recorded from guinea-pig atrial myocytes, using the whole-cell configuration of patch-clamp method. KEY RESULTS: Bath application of phenylephrine enhanced I (Ks) concentration-dependently with EC(50) of 5.4 microM and the maximal response (97.1+/-11.9% increase, n=16) was obtained at 30 microM. Prazosin (1 microM) almost totally abolished the potentiation of I (Ks) by phenylephrine, supporting the involvement of alpha(1)-adrenoceptors. The stimulatory action of phenylephrine was significantly, if not entirely, inhibited by the general PKC inhibitor bisindolylmaleimide I but was little affected by Gö-6976, Gö-6983 and rottlerin. Furthermore, this stimulatory effect was significantly reduced by dialyzing atrial myocytes with PKCepsilon-selective inhibitory peptide epsilonV1-2 but was not significantly affected by conventional PKC isoform-selective inhibitory peptide betaC2-4. Phorbol 12-myristate 13-acetate (PMA) at 100 nM substantially increased I (Ks) by 64.2+/-1.3% (n=6), which was also significantly attenuated by an internal dialysis with epsilonV1-2 but not with betaC2-4. CONCLUSIONS AND IMPLICATIONS: The present study provides experimental evidence to suggest that, in native guinea-pig cardiac myocytes, activation of PKC contributes to alpha(1)-adrenoceptor-mediated potentiation of I (Ks) and that epsilon is the isoform predominantly involved in this PKC action.

Silefrin, a sodefrin-like pheromone in the abdominal gland of the sword-tailed newt, Cynops ensicauda.

Sodefrin-like female-attracting pheromone was purified from the abdominal glands of male sword-tailed newts, Cynops ensicauda, by gel-filtration chromatography and reversed-phase high-performance liquid chromatography. The final product comprises 10 amino acid residues with the sequence SILSKDAQLK which coincided with the sequence deduced from its precursor cDNA. This peptide was designated silefrin. The sequence of silefrin was different from that of sodefrin by two amino acid residues, with substitutions Leu for Pro and Gln for Leu at positions 3 and 8, respectively. Both native and synthetic silefrin exerted an equipotent activity in attracting conspecific females.

Molecular cloning of newt sex pheromone precursor cDNAs: evidence for the existence of species-specific forms of pheromones.

Cloning of cDNA encoding a decapeptide pheromone (sodefrin) that attracts conspecific female newts was attempted. A cDNA clone encoding a protein consisting of 189 amino acid residues including a sodefrin sequence was isolated from a Cynops pyrrhogaster abdominal gland cDNA library. Likewise, a cDNA clone encoding a molecule comparable to the sodefrin precursor was obtained from a Cynops ensicauda abdominal gland cDNA library. This clone encoded a precursor protein of 192 amino acid residues, including a sodefrin-like peptide sequence with substitutions of two amino acid residues. This is the first report of a peptide pheromone precursor in vertebrates.

Female-attracting pheromone in newt cloacal glands.

It has been postulated that male newts emit pheromones that attract females of the same species. Female newts of the species Cynops pyrrhogaster were attracted to water in which sexually active conspecific males had been kept, but not to water in which abdominal gland-ablated males had been kept, indicating that the attracting pheromone was secreted by or through the abdominal gland of the cloaca. An attempt has been made to isolate and characterize the female-attracting pheromone in the abdominal glands of male newts. Female-attracting activity was monitored using a preference test. The active substance was isolated by two steps of purification using reverse-phase high-performance liquid chromatography. Direct sequencing of the final product revealed that it is a decapeptide with the amino acid sequence Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys. Its minimum effective concentration was estimated to be between 0.1 pM and 1.0 pM. The synthetic peptide showed a female-attracting activity similar to that of the native peptide. It seems to act through the olfactory organ of female newts, because the effect of the peptide was blocked by bilateral nostril plugging with cotton balls soaked in melted vaseline. An antiserum against sodefrin was generated in a rabbit. An immunoelectron microscopic study using this antiserum revealed that sodefrin exists predominantly within secretory granules in the epithelial cells of the abdominal glands. A radioimmunoassay for sodefrin was developed in which the antiserum was used along with sodefrin which was N-terminally extended with a tyrosine residue as a radioligand. The immunoassayable sodefrin content in C. pyrrhogaster males was diminished by castration and hypophysectomy. The sodefrin content was increased markedly in the castrated and hypophysectomized newts after treatment with both testosterone and prolactin. Testosterone but not prolactin increased the sodefrin content to a lesser extent. Aqueous extract of the abdominal glands of C. ensicauda showed no inhibition of binding in this assay. Moreover, C. ensicauda females were insensitive to sodefrin, although they were attracted to a water extract of abdominal glands from males of their own species. On the other hand, C. pyrrhogaster females responding to sodefrin were not attracted to the water extract of the abdominal glands from C. ensicauda males. Sequence analyses of sodefrin cDNA clones obtained from a C. ensicauda abdominal gland cDNA library revealed that the cDNA encoded a variant type of sodefrin peptide with substitutions of Leu3 and Gln8. The synthetic [Leu3, Gln8]-sodefrin attracted C. ensicauda females but not C. pyrrhogaster females. These results indicate that the female-attracting pheromone differs between these two species of genus Cynops.

Hormonal control of response to and secretion of sex attractants in Japanese newts.

Hormonal control of sexual chemoattraction in the newt, Cynops pyrrhogaster, was investigated by a water preference test. Sexually inert newts injected with prolactin (PRL) and human chorionic gonadotropin (HCG) in combination preferred the water in which newts of the opposite sex had been kept, whereas saline-injected specimens did not. This indicates that PRL and HCG enhance the response of the newts to attractant(s) secreted by the opposite sex. The water in which PRL plus HCG-treated newts had been kept attracted the opposite sex more intensely than the water in which PRL-, HCG-, or saline-injected newts had been kept. In castrated males, PRL plus testosterone propionate (TP), and in ovariectomized females, PRL plus estradiol induced the secretion of substance(s) that attracted newts of the opposite sex. Removal of the abdominal gland of the cloaca in the male or the oviduct in the female attenuated the attracting activity of the water in which they were kept. Involvement of PRL and gonadal steroids in the secretion of sex attractants by the male abdominal gland and female oviduct was suggested.

Endocrine regulation of reproductive behavior in the newt Cynops pyrrhogaster.

Hormonal control of the expression of courtship behavior and of secretion of the female-attracting pheromone sodefrin by the male red-bellied newt, Cynops pyrrhogaster, together with the hormonal influence on the responsiveness to the pheromone in the female, is reviewed.Expression of the initial stage of the courtship behavior, i.e., tail vibration by the male in front of the female, is dependent on prolactin (PRL) and androgen. During the courtship, sodefrin seems to be released from the cloaca through the ducts of the abdominal gland. Both content of immunoreactive sodefrin and preprosodefrin mRNA levels in the abdominal gland are elevated by a combination of PRL and androgen, indicating that the pheromone synthesis is stimulated by these two hormones. On the other hand, the discharge of sodefrin is accelerated by AVT, its action being mediated by V1 receptor. In female newts, responsiveness of the vomeronasal epithelium to the pheromone is elevated by a combination of PRL and estrogen. Thus, it can be concluded that PRL, AVT, and sex steroids are key hormones for the reproductive performance in the red-bellied newt. In this article, the significance of the structure of the pheromone molecule as a peptide is also discussed in terms of its species-specificity and its effectiveness in an aquatic environment.

Hormonal influence on the olfactory response to a female-attracting pheromone, sodefrin, in the newt, Cynops pyrrhogaster.

Sodefrin is a female-attracting pheromone isolated from the abdominal glands of male newts, Cynops pyrrhogaster. Previously, the preference of conspecific female newts for sodefrin was shown to be completely abolished by plugging the bilateral nostrils, indicating that it acts on the olfactory organ. To determine the sensitivity of the olfactory receptor cells to sodefrin, electro-olfactograms (EOGs) in response to sodefrin solution were recorded from the ventral nasal epithelium of sexually developed female newts. Sodefrin elicited marked EOG responses in a dose-dependent manner on the epithelium of the lateral nasal sinus (LNS) region, a putative vomeronasal organ. In ovariectomized females, treatment with prolactin (PRL) and estrogen markedly enhanced the EOG response to sodefrin. The EOG response to the pheromone was also enhanced considerably by treatment with either PRL or estrogen alone. A slight but significant elevation was observed in castrated males receiving PRL plus estrogen or estrogen alone. It was concluded that the main site of action of sodefrin resides in the lateral sinus region and that sensitivity to sodefrin is under the control of PRL and estrogen. The presence of a sex difference in olfactory sensitivity to the hormones and/or pheromone was also suggested.

Hormonal control of urodele reproductive behavior.

Hormonal control of expression of courtship behavior and of development of structures related to the reproductive behavior in two species of Japanese newts, Cynops pyrrhogaster and Cynops ensicauda, was described. Prolactin (PRL) and androgen were essential factors for eliciting courtship behavior. In addition, arginine vasotocin markedly enhanced the expression of courtship behavior. PRL induced migration to water, in which courtship and oviposition take place, and converted the integument from the terrestrial type to the aquatic one. PRL also stimulated the growth of the tail fin, which was blocked by estrogen. Cellular and nuclear size and number of synapses on the somata of Mauthner cells, which are involved in tail movement, were also increased by PRL and androgen. Synthesis of sodefrin, a female-attracting pheromone, in the abdominal gland as well as that of mucopolysaccharides constituting the sac of sperm in the lateral gland was enhanced by PRL and androgen. Structural development of oviducts was elicited by estrogen or PRL to a certain extent, and full oviducal development by the combination of these two hormones, PRL being indispensable for the oviducal jelly secretion.

[Epidural injection for postoperative pain relief after thoracoscopic surgery].

For the pain relief after thoracoscopic surgery, the epidural injection of buprenorphine was performed in 46 cases. In 28 cases, intermittent injection was performed before awaking from anesthesia. The effect was excellent in 6 cases, who were free from pain without any more injection after returning to the ward, and good in 27 cases, who sometimes felt dull pain and had another analgetics (intermittent epidural injection in 16 cases, intermittent epidural injection and another medication in 5 cases). In 18 cases, continuous injection was performed. The effect was excellent in 9 cases and good in 9 cases. It was concluded that the epidural injection had effective analgesic effect after thoracoscopic surgery, both in intermittent injection group and continuous injection group. For limited medication, the intermittent epidural injection was considered the first choice after thoracoscopic surgery.

[An elderly case of pneumothorax treated with omentopexy].

A 74-year-old male was admitted to our hospital because of left pneumothorax with persistent air leakage. He had undergone negative pressure drainage, chemical pleurodesis and transbronchial embolization in another hospital. Chest X-ray and CT scan showed collapse of the left lung and a defect of the pleura in the left lung S9. Patch closure was performed for the round pleurobronchial fistula (35 x 35 mm in size) using polyglycol acid felt and fibrin glue. But as severe air leakage was observed again about 24 hours after surgery, omentopexy was performed. The postoperative course was uneventful, and he was discharged 17 days after the second surgery.

[A case of adrenal myelolipoma].

Adrenal myelolipoma is a rare, nonfunctioning, and benign tumor composed of varying proportion of fat and bone marrow elements. A case is presented of a thirty-six-year-old woman in whom the diagnosis of adrenal myelolipoma was suspected preoperatively by ultrasonography, computed tomography, and magnetic resonance imaging (MRI). The patient cured by surgery. Histological examination showed mature adipose tissue and normal hemopoietic tissue. This is the first report which describes MRI of adrenal myelolipoma in Japan.

Sodefrin: a novel sex pheromone in a newt.

The abdominal gland in the male red-bellied newt, Cynops pyrrhogaster, is the source of a female-attracting pheromone. An attempt was made to isolate and characterize the female-attracting pheromone in the abdominal glands of male newts. The active substance, named sodefrin (from the Japanese 'sodefuri' which means 'soliciting') has been isolated and shown to be a novel decapeptide with the sequence, Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys. Its minimum effective concentration in water is 0.1-1.0 pmol 1-1. Synthetic sodefrin shows a female-attracting activity similar to that of the native peptide, and acts through the olfactory organ of female newts. Electrophysiological studies reveal that sodefrin evokes a marked electroolfactogram response in the vomeronasal epithelium in sexually mature females and in ovariectomized females treated with prolactin and oestrogen. The pheromonal activity of sodefrin appears to be species-specific since it does not attract females of a congeneric species, the sword-tailed newt C. ensicauda. However, C. ensicauda has a variant of sodefrin differing from that in C. pyrrhogaster by substitutions of Leu for Pro at position 3 and Gln for Leu at position 8. The C. ensicauda variant sodefrin does not attract C. pyrrhogaster females. Genes encoding the sodefrin precursor protein have been cloned in both C. pyrrhogaster and C. ensicauda. Immunostaining of the abdominal gland using the antiserum against sodefrin shows that sodefrin occurs in the epithelial cells, predominantly within the secretory granules. Sodefrin content, detected by immunoassay, in C. pyrrhogaster males decreases after castration and hypophysectomy and increases markedly in the castrated and hypophysectomized newts after treatment with androgen and prolactin. This combination of hormones also enhances sodefrin mRNA content in the abdominal gland as assessed by northern blot analysis using sodefrin cDNA.

Hormonal induction of male courtship behavior in the Japanese newt, Cynops pyrrhogaster.

In the breeding season, the sexually mature male newt, Cynops pyrrhogaster, vibrates the tail in front of the female at an early stage of courtship. Effects of prolactin (PRL), gonadotropin (GTH), and sex steroids on this behavior were investigated in the male paired with the female receiving PRL and GTH. The behavior was elicited in the sexually inert male by injections of PRL of bovine, ovine, or bullfrog origin and human chorionic gonadotropin or bullfrog LH and FSH in combination. The effect of PRL or GTH alone was less marked than that of PRL plus GTH, especially in terms of frequency of the behavior. In the hypophysectomized male, combination of PRL and GTH significantly increased both the incidence and frequency of the behavior. However, PRL alone was not effective, and the effect of GTH alone was less pronounced than that in the intact animal receiving GTH injections. The effect of GTH was nullified by castration. In the PRL-treated castrated animal, testosterone or dihydrotestosterone, but not estradiol, was effective in inducing the behavior.

Involvement of endogenous prolactin in the expression of courtship behavior in the newt, Cynops pyrrhogaster.

In order to ascertain the involvement of endogenous prolactin (PRL) in the expression of courtship behavior by the male newt, Cynops pyrrhogaster, antiserum against homologous PRL was administered to male newts, which had been exhibiting courtship behavior in the field and had been taken to the laboratory. The effect of the antiserum on the preference of terrestrial over aquatic habitats also was investigated. Within 24 hr of the first injection of antiserum, both incidence and frequency of courtship behavior declined markedly compared with those in the preimmune serum-injected newts. The anti-PRL serum-induced decline became more conspicuous thereafter. These animals came to prefer a terrestrial habitat. Cessation of antiserum injection and administration of PRL restored the expression of courtship behavior to a certain extent and altered their habitat preference from terrestrial to aquatic. The results strongly suggest the involvement of endogenous PRL in the expression of courtship behavior as well as in the preference of aquatic habitat.

Involvement of arginine vasotocin in reproductive events in the male newt Cynops pyrrhogaster.

Effects of arginine vasotocin (AVT) on reproductive events such as courtship behavior, pheromone release, and spermatophore discharge were investigated in the male newt Cynops pyrrhogaster. AVT enhanced the incidence and frequency of androgen-induced courtship behavior. In this case, AVT was likely to act centrally because the behavior was evoked with a much smaller amount of AVT when the hormone was administered intracerebroventricularly than when given intraperitoneally. Involvement of endogenous AVT in spontaneously occurring courtship behavior was also evidenced by the fact that administration of a V1 (vasopressor) receptor antagonist, [d(CH2)5(1), Tyr(Me)2, Arg8-vasopressin] suppressed the expression of the courtship behavior. The water in which AVT-treated males had been kept showed considerable female-attracting activity as compared with the water in which saline-injected males had been kept. Moreover, the content of sodefrin, a female-attracting pheromone in the abdominal gland, was decreased by the intraperitoneal injection of AVT, suggesting that the neurohypophyseal hormone stimulated the release of sodefrin from the abdominal gland into the water. AVT induced contraction of the excised abdominal gland concentration-dependently, and, again, the V1 receptor antagonist suppressed the AVT-induced contraction. Thus, we concluded that AVT induces the pheromone discharge, acting peripherally on a contractile structure of the abdominal gland. AVT was also found to induce spermatophore deposition in the male kept in the absence of the female. Administration of the V1 receptor blocker to the sexually developed males suppressed the spermatophore deposition. All these results indicate the involvement of AVT in reproductive events acting centrally and peripherally.

[New sex pheromone isolated from the abdominal glands of male newt, Cynops pyrrhogaster]. / Une nouvelle phéromone sexuelle isolée à partir des glandes abdominales du Triton mâle, Cynops pyrrhogaster.

Sodefrin, a novel decapeptide which attracts female newts, Cynops pyrrhogaster was isolated from the abdominal gland of the male of the same species. Synthetic sodefrin was tested for its activity with sexually active females and males and sexually inert females. It attracted only sexually active females. The effect of sodefrin was blocked by a bilateral nostril plugging with cotton balls soaked in melted vaseline. Immunoelectronmicroscopic study using antiserum against sodefrin revealed that sodefrin was located mainly in the secretory granules in the epithelial cells of the abdominal gland of the cloaca.

Radioimmunoassay of a newt sex pheromone, sodefrin, and the influence of hormones on its level in the abdominal gland.

A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a female-attracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its C-terminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the sword-tailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 +/- 3.4 pg/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormone-treated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.

Cold acclimation of guinea pig depressed contraction of cardiac papillary muscle.

Guinea pigs were exposed to 5 degrees C for 3 wk, and the contractions of myocardial papillary muscle were compared with preparations dissected from control animals kept at approximately 25 degrees C. Developed tension of the papillary muscle per cross-sectional area was significantly (t-test, P < 0.05) decreased after cold exposure (19,200 +/- 8,160 vs. 3,020 +/- 2,890 dyne/cm2; 1 Hz). Time to peak tension was significantly faster in cold-exposed guinea pigs (126.4 +/- 11.1 ms; 1 Hz) than in controls (162.7 +/- 8. 7 ms). The magnitude of the developed tension after application of ryanodine (2 mM) to muscles from cold-exposed animals was decreased to 37.5 +/- 8.3% of control at 1 Hz, whereas in muscles from control animals, tension was decreased to 82.4 +/- 7.7%. The ryanodine-sensitive component of contraction was not significantly changed in control guinea pigs at frequencies >0.5 Hz, whereas in muscles from cold-acclimated guinea pigs, there was a "positive staircase." These results suggested that reversal of the Na+/Ca2+ exchanger is predominantly involved in the positive staircase in control guinea pigs, whereas rate-dependent increases in the Ca2+ store in the sarcoplasmic reticulum may be involved in the staircase after cold acclimation.