Variable alteration of regional tissue oxygen pressure in rat hippocampus by acute swimming exercise.

UNLABELLED: One of the events in the brain is an increasing cerebral blood flow during exercise. The tissue oxygen level may be increased because blood flow correlates with tissue oxygen level. However, it is little known whether the tissue oxygen pressure in hippocampal region (Hip-pO2) will be affected by exercise. AIMS: The aim of this study is to examine Hip-pO2 levels in the hippocampus and its changes during exercise. MAIN METHODS: We applied improved Clark-type electrodes to measure Hip-pO2 level in the hippocampus of rats that were subjected to three groups, 2h swimming without weights (low intensity, n=6), 2h swimming with a 5 g weight (moderate intensity, n=6), and 2h swimming with a 10 g weight (high intensity, n=6). KEY FINDINGS: Exercise affected the Hip-pO2 level, the responses varied with the exercise intensity and duration. Interestingly during and after the Low intensity swimming the Hip-pO2 level showed long lasting enhancement (10-20% above resting level). But the moderate and high intensity swimming increased Hip-pO2 level at the start of the swimming (50%, P<0.05 and slightly above resting level, respectively, at 10 min of 2h swimming) and then began to decrease (at 120 min and 10 min of 2h swimming, respectively), and suppressed the Hip-pO2 levels during post exercise resting period (2h) (85-95% of resting level, NS and 60-70% of resting level P<0.05, respectively). SIGNIFICANCE: We propose that exercise-induced hippocampal hyper/hypo oxygen condition may participate in beneficial exercise effects on brain function.

Glycogen depletion in intrafusal fibres in rats during short-duration high-intensity treadmill running.

AIM: The recruitment patterns of the intrafusal and extrafusal fibres in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats were investigated during brief-intensity exercise by assaying their glycogen content histochemically. METHODS: Six adult male rats were assigned to each of four groups that ran up a 6 degrees incline on a motor-driven treadmill, at 40 m min(-1) for either 0.5, 1, 2, or 4 min. Six adult male rats in the control group did not run. Extrafusal and intrafusal fibres were classified by myosin ATPase staining. Optical densities for glycogen content were evaluated in serial periodic acid Schiff (PAS) stained-sections from the B and C regions of intrafusal fibres. RESULTS: The glycogen content of type IIA fibres in the SOL and EDL muscles decreased significantly in the early phase of exercise whereas the glycogen content of type I fibres in these muscles decreased later than that of type IIA fibres. The glycogen content of bag2 fibres decreased after 1 min of exercise in the SOL muscle and after 2 min of exercise in the EDL muscle. On the other hand, the glycogen content of bag1 and chain fibres decreased significantly after 2 min in the SOL muscle but not in the EDL muscle. CONCLUSION: The results suggest that during brief-intensity exercise, as the glycogen content of type IIA fibres is reduced earlier than that of type I fibres, bag2 fibres are most important early in this type of exercise.

A biphasic regulation of receptor mRNA expressions for growth hormone, glucocorticoid and mineralocorticoid in the rat dentate gyrus during acute stress.

Acute stress increases circulating ACTH and glucocorticoid levels. The hippocampus (HIP) is a target of such stress hormones as glucocorticoid and it also expresses receptors for growth hormone (GH), particularly in the dentate gyms (DG). In order to understand the interactions between glucocorticoids and functions of GH in HIP during acute stress, the mRNA levels for GH receptor (GHR), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were investigated in DG in rats exposed to restraint stress in the water (RSW). Using in situ hybridization histochemistry (ISHH), high level expressions of GHR mRNA were detected in DG. These were down-regulated by 14% after 0.5 h of RSW and then up-regulated by 38% over the initial level after 4 h of RSW. This biphasic enhancement of GHR mRNA expression in DG followed the elevation of plasma glucocorticoid levels and paralleled with biphasic expressions of mRNAs for GR and MR in DG. Although circulating GH levels did not show any correlation with the hippocampal GHR mRNA expression, adrenalectomy (ADX) decreased GHR mRNA expression in DG, and the dexamethasone treatment (DEX; 20 microg/100 microl, i.p.) of ADX rats rapidly increased the GHR mRNA expression in DG. These results have suggested that the GHR mRNA expression in the DG is regulated, at least in part, by glucocorticoids and that GH may be involved in responses of the DG to acute stress.

20 kDa human growth hormone (20K hGH) stimulates insulin-like growth factor-I (IGF-I) gene expression at lower concentrations than 22K hGH in hGH receptor-expressing Ba/F3 cells.

Growth hormone (GH) secreted from the pituitary is essential for postnatal growth in animals. GH exerts its actions by a direct effect on target organs and by stimulating insulin-like growth factor I (IGF-I) production. In the human pituitary, there is a naturally occurring variant protein which has a molecular mass of 20 kDa (20K hGH) besides the major 22 kDa hGH (22K hGH), but the physiological actions of 20K hGH are still poorly understood. In this study we have examined its effects on the IGF-I mRNA expression in the pro B-cell line Ba/F3 cells stably expressing hGH receptor (Ba/F3-hGHR). Ba/F3-hGHR cells were incubated for 2 h with a series of various concentrations (10 pM to approximately 10 nM) of 20K or 22K hGH. The IGF-I mRNA expression in the Ba/F3-hGHR cells was detected by the RT-PCR method. IGF-I gene expression was increased by 20K and 22K hGH stimulation, but not by PRL or IL-3 in the Ba/F3-hGHR. And this effect was not observed in parental Ba/F3 cells. Lower concentrations of 20K hGH more strongly induced IGF-I gene expression than 22K-hGH. These results suggest that 20K and 22K hGH stimulate the IGF-I gene expression in the Ba/F3-hGHR through hGH receptors, and that the stronger effect of 20K hGH than that of 22K hGH in enhancing the IGF-I gene expression may be correlated with a 20K hGH specific receptor dimerization mechanism.

Subcutaneously administered prolactin and 20K hGH, but not rGH or 22K hGH, prevent restraint stress-induced gastric ulcers in rats.

Stress causes gastric ulcer in vertebrates. In humans, growth hormone (hGH) and prolactin (hRPL) are promptly released into the circulation under the stress conditions, while in rats exposed to stress, the circulating levels of GH (rGH) are decreased and the circulating PRL (rPRL) levels are rapidly increased as in humans during stress. However, the roles of the circulating rGH and rPRL during stress are still unclear. Here we analyzed whether 22K hGH, 20K hGH or rGH, when compared to rPRL, can affect restraint stress in water (RSW)-induced gastric ulcers. Pretreatments of rats with subcutaneously (s.c.) administered rPRL or 20K hGH clearly prevented the development of the gastric injuries in rats subjected to 7 h RSW. The s.c. pretreatment with 22K hGH resulted in little cytoprotection in the rats exposed to RSW, while s.c. pretreatment with rGH showed no such protective effect against RSW-induced gastric injuries. Results suggested that rPRL and 20K hGH were acting on PRL receptor, but not on GH receptor, to prevent RSW-induced gastric injuries.

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities.

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.

Decreased expression of the mRNA for somatostatin in the periventricular nucleus of depression-model rats.

Expression of the mRNA for somatostatin (SRIF) in the periventricular nucleus (PeN), the level of SRIF in the stalk-median eminence (SME) and the concentration of growth hormone (GH) in the plasma were examined in depression-model rats in an attempt to confirm the hypothesis that SRIF neurons in the hypothalamus are hypofunctional in this model. We exposed male Wistar rats to intermittent walking stress for two weeks and then we measured their spontaneous running activity for 12 days. We divided the rats into a depression-model group and a partial-recovery group according to the spontaneous running activity of each rat after the termination of exposure to stress. Expression of SRIF mRNA in the PeN of the hypothalamus was monitored by in situ hybridization and relative levels were determined with an image analysis system. The relative level of expression of SRIF mRNA in the PeN was lower in rats in the depression-model group than in the control group and the partial-recovery group. The level of SRIF in the SME was lower and the plasma concentration of GH was higher in the depression-model group than in the other groups. Our findings suggest that reduced expression of mRNA for SRIF in the PeN might be associated with the pathophysiology of rats with this particular model of depression.

Expression and purification of a mutant human growth hormone that is resistant to proteolytic cleavage by thrombin, plasmin and human plasma in vitro.

The region having a sequence from amino acid 134 to 150 in human growth hormone (hGH) is known to be cleaved by proteases in human plasma, plasmin and thrombin. In this study, oligonucleotide primer-directed mutagenesis was used to produce recombinant mutant hGHs resistant to limited proteolysis by these proteases. Substitution of Arg134 and Thr135 of hGH with Asp134 and Pro135 yielded a thrombin-resistant hGH mutant, and substitution of Arg134, Thr135 and Lys140 with Asp134, Pro135 and Ala140 yielded a plasmin-resistant hGH mutant. The latter mutant hGH was also insensitive to in vitro proteolysis by human plasma incubated for 7 days. These alterations in amino acid residues of hGH did not disrupt its biological conformation and retained full growth promoting activities on rat Nb2 cells and human T-47D breast cancer cells.

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission.

Mice lacking expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme of the catecholamine biosynthetic pathway, in dopaminergic neuronal cell types were generated by a transgenic rescue approach to clarify the role of dopamine signaling during postnatal development. Introduction of the TH transgene directed by the dopamine beta-hydroxylase gene promoter into TH knockout mice restored noradrenaline and adrenaline synthesis, preventing perinatal lethality and cardiac dysfunction in the knockout mice. Lack of TH expression in the cells that normally express the dopaminergic phenotype resulted in a marked reduction of dopamine accumulation in the tissues, which led to multiple behavioral abnormalities at the juvenile stage. These abnormalities were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine-induced hyperactivity, cataleptic behavior, and defects in active avoidance learning. In contrast, development of the pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained, despite defective dopamine synthesis. These results demonstrate that dopamine neurotransmission is essential for controlling spontaneous and voluntary movement and associative learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways.

Enhanced response of growth hormone to growth hormone-releasing hormone and a decreased content of hypothalamic somatostatin in a stress-induced rat model of depression.

This study was designed to evaluate changes in the hypothalamic somatostatin-growth hormone axis (SRIF-GH axis) in a stress-induced rat model of depression. We exposed male Wistar rats to intermittent walking stress for two weeks, and then measured their spontaneous running activities for 12 days. We divided the rats into the depression-model group and the partial recovery group according to their spontaneous running activities after the termination of exposure to stress. We examined the secretion of GH from the anterior pituitary by injecting human GH-releasing hormone (hGHRH) with intracardiac cannulae or by applying hGHRH or SRIF to isolated anterior pituitaries using a perifusion system. We also determined SRIF content in the stalk-median eminence (SME) and the plasma concentration of GH. In the depression-model group, intracardiac administration of hGHRH caused the enhanced release of GH into plasma, while application of hGHRH or SRIF to the anterior pituitary in vitro had similar effects on GH release in the control and partial recovery groups. Furthermore, the SRIF content was decreased in the SME and the GH concentration was increased in plasma. The partial recovery group gave similar values to the control group. The enhanced response of GH to hGHRH in the depression-model group might have been caused by the reduced content of SRIF in the SME in view of the unchanged response of GH to the infusion of hGHRH or SRIF in the perifusion system.

The growth hormone (GH) gene is expressed in the lateral hypothalamus: enhancement by GH-releasing hormone and repression by restraint stress.

Recent studies suggest that GH may modulate emotion, behavior, or stress response by its direct actions on the brain, and possible expression of the GH gene in the brain has been predicted. In this study we have investigated whether and where the GH gene is expressed in the brain and how it is regulated. Ribonuclease protection assay and 5'-rapid amplification of complementary DNA ends-PCR analyses indicated that the GH gene was expressed in rat brain, initiating at the identical transcription start point as that for pituitary GH gene expression. The brain GH messenger RNA was predominantly detected in the lateral hypothalamus (lh) by in situ reverse transcription-PCR analysis. GH gene expression in the brain was significantly enhanced by GH-releasing hormone administration and was rapidly repressed by exposure to restraint stress in the water, whereas the changes in pituitary GH messenger RNA contents in these circumstances were relatively smaller. The results of the present study suggest that the brain GH is predominantly expressed in lh under the control of physiological conditions to play a role in the modulation of brain functions.

Restraint stress enhances the gene expression of prolactin receptor long form at the choroid plexus.

Hormonal control of brain functions is considered to be important in the tolerance of stress, and it is now established that stress elevates serum PRL levels in male or cycling female rats. To investigate whether or how serum PRL acts on the brain during exposure to stress, we analyzed serum PRL levels and the gene expression of brain PRL receptors in rats subjected to restraint stress in the water (RSW). The serum PRL concentration was remarkably increased within 30 min in the rats by exposure to RSW and decreased to the initial level after 4 h of RSW, remaining at this level for up to 7 h of RSW. After the rats were released from the stress, the serum PRL level was significantly lowered in 6 h. Ribonuclease protection assay and in situ hybridization analysis revealed that messenger RNA (mRNA) expression for the long form PRL receptor [PRL-R(L)] was remarkably induced in the rat choroid plexus in 2 h of RSW. The high expression level of PRL-R(L) mRNA in the region was reduced after the rats were released from the stress. PRL-R(L) mRNA expression in the hypothalamus was at lower levels than those in the choroid plexus before and during the RSW treatment. The short form PRL receptor mRNA expression in the rat brain was considerably lower than expression of the long form receptor mRNA before or during RSW. The results indicated that the restraint stress caused a rapid increase in serum PRL and induced the gene expression for PRL-R(L) in the choroid plexus, suggesting stress-induced and choroid plexus PRL-R(L)-mediated transport of serum PRL into the cerebrospinal fluid.