Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats

Both trigeminal and spinal ganglion neurons show a strong potentiation of responses to the irritant capsaicin in an acidic environment. The present study revealed that there is also a strong interaction between protons and piperine, another vanilloid irritant. We studied the mechanism of the interaction between protons and piperine. Whole-cell patch clamp recordings were performed on cultured adult rat trigeminal ganglion (TG) neurons voltage-clamped near their resting membrane potential (-60 mV). Piperine (10 microM) caused a sustained net inward current associated with either an increase or decrease in membrane conductance. When protons and piperine were co-applied, the membrane currents evoked in piperine-sensitive TG neurons far exceeded the algebraic sum of the responses to the two stimuli applied in isolation. Capsazepine blocked the response of TG neurons to piperine at both physiological and acidic pH. In the presence of capsazepine, responses to the mixture of piperine and protons resembled the response to the low pH stimulus applied alone. Capsazepine had no effect on the sustained proton-induced current. These findings suggest that protons enhance the piperine current by altering the vanilloid receptor/channel complex or increasing the length constant of the space clamp.     

Effects of growth hormone secretagogues on prolactin release in anesthetized dwarf (dw/dw) rats

In addition to stimulating GH release, GH secretagogues such as GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH and PRL release. Although the effects on ACTH have recently been studied, there is little information about the effects of GHRP-6 on PRL. We have now studied GHRP-6-induced GH and PRL release and their regulation by estrogen (E2) in anesthetized male and female rats and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1-2.5 microg, i.v.) did not induce PRL release in normal male or female rats, significant PRL responses were observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2 treatment. These effects could be dissociated from GHRP-6-induced GH release in the same animals, but not from PRL release induced by TRH, which was also abolished by ovariectomy and induced in males by E2 treatment. However, the effects of GHRP-6 on PRL were unlikely to be mediated by TRH because in the same animals, TSH levels were unaffected by GHRP-6 whereas they were increased by TRH. The increased PRL response could reflect an increase in GH secretagogue receptor expression that was observed in the arcuate and ventromedial nuclei of E2-treated rats. Our results suggest that the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites.     

Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms

BACKGROUND & AIMS: Calpain proteases have been implicated in cell death by necrosis and more recently by apoptosis. Experiments were designed to determine the role of calpain proteases in ischemic rat liver injury by measurement of cytosolic calpain activity after different periods of ischemia-reperfusion and by evaluation of the effects of calpain inhibition on tissue injury and animal survival. METHODS: Calpain activity was measured in the cytosol using Suc-Leu-Leu-Val-Try-7 amino-4 methyl coumarin, a specific fluorogenic substrate, and Cbz-Leu-Leu-Tyr-CHN2, a specific inhibitor. RESULTS: Calpain activity increased significantly with the duration of ischemia-reperfusion and was inhibited more than 80% by the inhibitor. Calpain inhibition resulted in a significant decrease in transaminase release and tissue necrosis and converted nonsurvival ischemic conditions to survival conditions. When the in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling assay for apoptosis was used, 35% +/- 6% of nonparenchymal cells and 16% +/- 3% of hepatocytes stained positively after 60 minutes of ischemia and 6 hours of reperfusion. In contrast, animals pretreated with the calpain inhibitor showed minimal evidence of apoptosis. This was further substantiated by gel electrophoresis assay for DNA fragmentation and by electron-microscopic evaluation. CONCLUSIONS: These data suggest that calpain proteases play a pivotal role in warm ischemia-reperfusion injury of the rat liver through modulation of apoptosis and necrosis.     

Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo

Recent pharmacological and biochemical studies have suggested that there may be more than one molecular form of the neurokinin-1 receptor (NK-1), a long and short isoform differing in the length of their cytoplasmic carboxyl-terminal tails, but no definitive evidence of the existence of such NK-1 receptor isoforms in tissue has been presented. To examine whether these different isoforms are expressed in vivo we have compared the distribution of high affinity substance P (SP) binding sites (visualized by autoradiography with [125I]SP), with the distribution of the C-terminal epitope of the full length receptor (visualized with a specific antibody against the extreme C-terminal sequence). The former method labels both long and short forms of the NK-1 receptor, while the latter labels only the long form of the protein. In the rat there is a close correspondence of [125I]SP binding and NK-1 immunoreactivity in the striatum, suggesting that the long isoform predominates in this tissue. In the parotid and submaxillary gland, there are very high levels of [125I]SP binding but only low levels of NK-1 immunoreactivity, suggesting that expression of the short form predominates in these tissues. These results imply that different tissues express different ratios of the two isoforms of the NK-1 receptor. This differential expression provides the theoretical basis for tissue specific pharmacological targeting of NK-1 receptors.     

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst2(b) receptor (CHOsst2(b)) and compared these to their effects in cells expressing the sst2(a) receptor (CHOsst2(a)). In contrast to the sst2(a) receptor, the sst2(b) receptor did not mediate inhibition of bFGF (10 ng ml(-1))-stimulated re-growth and cell proliferation. Rather, SRIF (0.1-1000 nM) and angiopeptin (0.1-1000 nM) stimulated basal re-growth and proliferation of CHOsst2(b) cells in a concentration-dependent manner (estimated pEC50 values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst2 receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst2(a) and sst2(b) receptor splice variants mediate opposite effects on cell proliferation.     

Heritabilities and genetic correlations in related dwarf and normal broiler populations

Heritability estimates were higher for 8-week body weight in dwarf than in normal broiler populations due to the maternal effects of dwarf dams. 2. The dwarfing gene dw did not induce new genetic variability for egg weight, 30-week body weight and age at sexual maturation. 3. Genetic correlation estimates showed that the selection for 8-week body weight will increase egg weight in dwarf pullets more than in normals. 4. Within a dwarf population it should be possible to increase 8-week body weight without influencing the mature size of pullets.     

Metabotropic glutamate receptor in GHRH and beta-endorphin neurones of the hypothalamic arcuate nucleus

Growth hormone-releasing hormone (GHRH) and beta-endorphin are mainly synthesized in neurones of the hypothalamic arcuate nucleus. Arcuate neurones also contain both ionotropic and metabotropic glutamate receptors. The aim of present study was to investigate whether glutamate receptors are present in GHRH and beta-endorphin containing nerve cells of this hypothalamic area. Using double-label immunocytochemistry as well as the mirror technique, we found that almost all GHRH and beta-endorphin immunoreactive arcuate neurones contain the metabotropic glutamate receptor la. The observations provide morphological evidence for the view that glutamate, which appears to be a major excitatory neurotransmitter in the hypothalamus, may directly stimulate GHRH and beta-endorphin neurones of the medial hypothalamus.     

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.     

Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say)

Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogenetic and triploid M. lateralis were successfully induced. For gynogenesis, eggs were fertilized with sperm irradiated with ultraviolet light and subsequently treated with cytochalasin B to block the release of the second polar body (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogenetic diploids were female, and there was no significant difference (P > 0.05) in sex ratio between diploids and triploids. These results suggested that the dwarf surfclam may have an XX-female, XY-male sex determination with Y-domination. Compared with diploids, triploids had a relative fecundity of 59% for females and 80% for males. Eggs produced by triploid females were 53% larger (P < 0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P > 0.33) different from normal diploid females, suggesting that inbreeding depression was minimal in meiosis II gynogens. Triploid clams were significantly larger (P < 0.001) than normal diploids. We hypothesize that the increased body-size in triploids was caused by a polyploid gigantism due to the increased cell volume and a lack of cell-number compensation.     

GABA(A) receptor function in the cerebral cortex of alcohol-naive P and NP rats

In our previous paper (Biochim. Biophys. Acta 1379 (1998) 257-263), we demonstrated that bicarbonate promotes a cleavage of lactone ring of dehydroascorbate (DHA) on the basis of in vitro experiments. In the present study, we examined the degradation of DHA in blood circulation in vivo by using a high-performance liquid chromatographic method for the determination of ascorbate (AsA), DHA and 2,3-diketogulonate (2,3-DKG), which required no pretreatment of biological fluids. When DHA was intravenously administered to rats, a rapid disappearance of DHA (t1/2 < 1 min) and a concomitant appearance of 2,3-DKG in blood circulation were observed. Approximately 90% of the administered DHA were excreted into urine as resulting 2,3-DKG (55%) and AsA (31%), respectively. Furthermore, we elucidated that rat plasma lacks an enzyme having an aldonolactonase-like activity. The result of the present study suggests that this DHA disappearance is a function of both a chemical degradation to 2,3-DKG and a reduction to AsA.     

Longtime administration of growth hormone-releasing hormone (GHRH) does not restore the reduced efficiency of GHRH on sleep endocrine activity in 2 old-aged subjects--a preliminary study

Aging results in a more shallow sleep accompanied by a blunted growth hormone (GH) secretion. In young male normal controls repetitive administration of GH-releasing hormone (GHRH) at the beginning of the night results in an increased secretion of GH, a blunting of cortisol and a stimulation of slow-wave sleep (SWS). In healthy elderly men and women, however, GHRH exerts only weak effects on sleep-endocrine activity. In a previous report continuous treatment of healthy elderly males by repetitive administration of GHRH (during 12 days administration with 100 micrograms GHRH i.v. at 9.00 h every second day, "priming") enhanced GHRH stimulated GH secretion at daytime markedly. We tested if priming with GHRH results in a more distinct modulation of the nocturnal hormone secretion and of the sleep EEG than acute administration of the peptide. Two elderly male controls spent first three consecutive nights in the sleep laboratory, the first of which served for adaptation to laboratory conditions. During the two other nights (at days 1 and 2) sleep EEG was recorded and blood was sampled for determining the secretion of GH, cortisol and ACTH. In one of the nights the subjects received 50 micrograms GHRH hourly between 22.00 h and 1.00 h (4 x 50 micrograms) or placebo. The next examination followed after the priming period at day 14 and the last was performed two weeks after treatment at day 28. After the baseline administration of 4 x 50 micrograms GHRH before priming no clear changes of sleep EEG towards improved sleep were detectable, whereas GH secretion was increased. After priming sleep period time and SWS time were lower compared to the baseline night with GHRH administration, whereas REM time duration increased. GHRH induced GH secretion was not enhanced after priming. ACTH secretion was markedly enhanced compared to baseline stimulation. We conclude that priming with GHRH has no sleep improving effect and does not change hormone secretion in elderly normal subjects. Hence in the elderly priming with GHRH is not capable to induce a rejuvenation of sleep endocrine activity.     

Evidence for receptor bound endothelin in renal but not in cardiac tissues from normal rats

The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 microM) and calmodulin (0.30 microM). These increases were clearly inhibited by the addition of regucalcin (0.50-1.0 microM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 microg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 microM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 microM) in the presence of Ca2+ (1 and 10 microM). This increase was completely inhibited by the presence of regucalcin (0.12 microM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 microM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin.     

Recombinant human erythropoietin (rhEPO) treatment potentiates growth hormone (GH) response to growth hormone releasing hormone (GHRH) stimulation in hemodialysis patients

On the basis of previously described effects of recombinant human erythropoietin (rhEPO) treatment on endocrine abnormalities present in uremia, we assessed the possible effect of treatment with rhEPO on growth hormone (GH) response to growth hormone releasing hormone (GHRH) in a group of uremic patients. Eight patients on maintenance hemodialysis for 12 to 228 months, not previously treated with rhEPO, were tested with 100 micrograms of GHRH i.v. in bolus before and after three months of rhEPO treatment (40 U/kg i.v. three times a week). Before treatment, the GH response to GHRH was characterized, in uremic patients, by remarkable differences in plasma GH values and in the pattern of response curve in single patients. The variability of GH response was not modified after rhEPO treatment; however, an overall potentiation of GH response with a significant increase of plasma GH (p = 0.017 at 15 min, p = 0.035 at 30 min after GHRH injection) was observed in the tests performed after treatment. rhEPO administration induced an evident improvement of anemia, blood hemoglobin concentration being 5.3-7.6 g/dl before and 9.1-11.3 g/dl after treatment; however a demonstrable correlation between the potentiation of GH response to GHRH and the increase of hemoglobin concentration was not observed.     

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.     

The role of the nerve growth factor carboxyl terminus in receptor binding and conformational stability

The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116-120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116-120, are important for both the structural stability and biological activity of NGF.