Seven Japanese Herbals Prolonged Cardiac Allograft Survival.

Japanese herbal medicines have long been used as alternative therapy because of their immunomodulatory effects. In recent years, use herbal medicines is rapidly increasing worldwide. In this study, we investigated the effect of 17 components of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA mice by using microsurgical techniques. Artemisiae capillaris herba (Inchinko) was given to CBA recipients at a dosage of 1 g/kg/day from the day of transplantation until 7 days afterward. The other 16 components were given at a dosage of 2 g/kg/day for the same time period. Naïve CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST] of 7 days). CBA transplant recipients given 2 g/kg/day of Glycyrrhizae radix (Kanzou), Poria sclerotium (Bukuryo), Pinellia tuber (Hange), Cnidii rhizome (Senkyu), Paeoniae radix (Shakuyaku), and Scutellariae radix (Ogon) had prolonged C57BL/6 allograft survival significantly (MSTs were 18, 18, 17, 14, 12, and 12 days, respectively). Moreover, CBA transplant recipients given 1g/kg/day of Artemisiae capillaris herba had prolonged C57BL/6 allograft survival (MST >100 days); however, none of other 10 components prolonged allograft survival. In conclusion, administration of 7 components of traditional Japanese herbal medicines might induce prolongation of fully major histocompatibility complex-mismatched cardiac allografts.

Induction of Regulatory CD4+ Cells and Prolongation of Fully Major Histocompatibility Complex Mismatched Murine Cardiac Allograft by Shigyakusan.

Shigyakusan (also known as Tsumura Japan [TJ]-35) is composed of peony, bitter orange, licorice, and Bupleuri radix is used for cholecystitis and gastritis as an anti-inflammatory agent. We investigated the effect of TJ-35 on alloimmune response in a murine heart transplantation model. CBA mice that underwent transplantation of a C57BL/6 (B6) heart were assigned to four groups: no treatment, TJ-35-exposed, each component-exposed, or each component missing-exposed. The four groups above each received oral administration of TJ-35, each component, or TJ-35 with each component missing from the day of transplantation until 7 days, respectively. Untreated CBA recipients rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). TJ-35-exposed CBA recipients had significantly prolonged B6 allograft survival (MST, 20.5 days). However, MSTs of CBA recipients that had been administered each component and TJ-35 with each component missing did not reach that of TJ-35-exposed recipients. Adoptive transfer of CD4+ splenocytes from TJ-35-exposed primary allograft recipients resulted in significant prolonged allograft survival in naïve secondary recipients (MST, 63 days). Flow cytometry studies showed that the percentage of CD4+CD25+Foxp3+ cell population was increased in TJ-35-exposed CBA recipients. In conclusion, TJ-35-induced prolongation of fully allogeneic cardiac allografts and may generate regulatory CD4+CD25+Foxp3+ cells in our model. The effect seemed to require all components of TJ-35.

Rikkunshito (TJ-43) Improved Reduction of Food Intake in a Murine Cardiac Transplantation Model.

Rikkunshito (TJ-43), an eight-component traditional Japanese herbal medicine, has been used in clinics for gastritis, vomiting, and appetite loss. We investigated the effects of TJ-43 on the amelioration of appetite loss in the surgical-exposed model of murine cardiac allograft transplantation. CBA mice underwent transplantation of a CBA (syngeneic group) or C57BL/6 heart (allogeneic group) and received oral administration of 2 g/kg/d of TJ-43 from the day of transplantation until 7 days afterward. The amount of food intake (FI) and weight change after operation were recorded from 1 to 28 postoperative days. The allogeneic group had less average amounts of FI for 1 week compared with the syngeneic group (FI was 1.90 ± 0.43 g and 2.66 ± 0.46 g, respectively). Average FIs between the syngeneic and allogeneic groups with TJ-43 for 1 week were 2.36 ± 0.44 g and 2.30 ± 0.13 g, respectively, and those with distilled water were 2.66 ± 0.46 g and 1.90 ± 0.43 g, respectively, suggesting that exposure with TJ-43 tended to ameliorate the reduction of FI. Similarly, the effect on the amelioration of average FI in syngeneic and allogeneic groups exposed for 2 weeks was confirmed. However, exposure to with TJ-43 had no effects on FI after 4 weeks. TJ-43 could prevent reduction of average FI induced by the surgical-exposed model of murine cardiac allograft transplantation.

Effect of 34 kinds of traditional Japanese herbal medicines on prolongation of cardiac allograft survival.

Herbal medicines have been used for over 3,000 years in Asian as alternative therapy for their variety effects and have recently become popular in Europe and the United States. In the last 30 years, Japanese herbal medicines were widely used for treatment of diseases after been recognized officially by Japanese government. In this study, we investigated the effect of 34 kinds of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. CBA mice (H2(k)) underwent transplantation of a C57BL/6 (H2(b)) heart and received oral administration of 2 g/kg/d of the 34 kinds of herbal medicines from the day of transplantation until 7 days afterward. Naïve CBA mice rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). CBA transplant recipients given 2 g/kg/d of Sairei-to (TJ-114) and Tokishakuyaku-san (TJ-23) had prolonged C57BL/6 allograft survival indefinitely (both MSTs > 100 days). Moreover, CBA transplant recipients given Seisinrensiin (TJ-111), Tokishigyakukagoshuyushokyoto (TJ-38), Rikkunshito (TJ-43), Maobushisaishinto (TJ-127), Ninjin-yoei-to (TJ-108), Ryokan-kyomi-shinge-nin-to (TJ-119), Inchingorei-san (TJ-117), Hochuekkito (TJ-41), Kihi-to (TJ-65), and Sinbu-to (TJ-30) had also prolonged C57BL/6 allograft survival significantly (MSTs of 28, 22, 16, 14, 14, 13, 12, 9.5, 9 and 9 days, respectively). However, none of other 22 kinds of herbal medicines could prolong the allograft survival. Furthermore, oral administration of 2 g/kg/d of Daikenchuto (TJ-100) induced sudden death (within 1 minute) in CBA mice. In conclusion, 12 kinds of Japanese herbal medicines prolonged allograft survival and one showed toxic effect in mice.

The smell of Tokishakuyaku-san (TJ-23) induces generation of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts in mice.

Oral administration of Tokishakuyaku-san (TJ-23), a Japanese herbal medicine, induces prolongation of cardiac allograft survival and generates regulatory cells in mice. Because herbal medicines usually have unique odor, and because smell is supposed to modulate the immune system, we examined whether the odor of TJ-23 induced prolonged allograft survival and regulatory cell generation. Naïve CBA mice (H2(k)) and olfactory-dysfunctional CBA mice after a stereotaxic operation underwent transplantation of C57BL/6 (B6, H2(b)) hearts, receiving fumigated water only or TJ-23 until rejection. Untreated or treated with water fumigation CBA mice rejected B6 cardiac grafts acutely (median survival times [MSTs], 7 and 8.5 days). When CBA mice were treated with fumigation of TJ-23, allograft survival was significantly prolonged (MST, 48 days). Olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 rejected grafts acutely (MST, 7 days). Treatment with fumigation of TJ-23 also suppressed splenocytes proliferation and interferon-γ production. Secondary CBA recipients of whole splenocytes or CD4(+) cells from primary TJ-23-treated CBA recipients of B6 cardiac allografts at 30 days after grafting showed prolonged survival of B6 hearts (MST, >60 days). Flow cytometry studies showed increased CD4(+)CD25(+)Foxp3(+) regulatory cells in recipients given fumigation of TJ-23. In conclusion naïve but not olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 displayed prolonged survival of fully allogeneic cardiac allografts and generation of regulatory cells.

Artemisiae capillaris herba induces prolonged survival of fully cardiac allografts and generates regulatory cells in mice.

Inchingorei-san (TJ-117), a 6-component herbal medicine, is used in Japan for the treatment of vomiting, urticaria, and liver and kidney disorders with few side effects. In this study we investigated the effect of TJ-117 on alloimmune responses in murine cardiac allograft transplantation. CBA (H2(k)) mice underwent transplantation of a C57BL/6 (B6, H2(b)) heart with oral administration of TJ-117 (or 1 component of TJ-117) from the day of transplantation for 7 days. CBA recipients given 1 g/kg/d of TJ-117 showed prolonged B6 allograft survival (median survival time [MST], 23 days). Naive CBA mice rejected B6 cardiac grafts acutely (MST, 7 days). Moreover, Artemisiae capillaris herba (ACH; 1g/kg/d) 1 component of TJ-117, significantly prolonged B6 allograft survival (MST, > 100 days). However, the other 5 components of TJ-117 were individually less effective than TJ-117 or ACH. ACH also suppressed splenocyte proliferation and interferon-γ production. Secondary CBA recipient showed prolonged survival of B6 hearts after treatments with whole splenocytes from primary ACH-treated CBA recipients carrying B6 cardiac allografts for 30 days (MST, >50 days). Flow cytometry studies showed increased CD4(+)CD25(+)Foxp3(+) regulatory cells in transplant recipients given ACH. In conclusion, ACH, 1 component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts with generation of CD4(+)CD25(+)Foxp3(+) regulatory cells.

Music exposure induced prolongation of cardiac allograft survival and generated regulatory CD4⁺ cells in mice.

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2(K)) underwent transplantation of a C57BL/6 (B6, H2(b)) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4(+) cells, and CD4(+)CD25(+) cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4(+)CD25(+)Foxp3(+) cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4(+)CD25(+)Foxp3(+) regulatory cells.

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5(+)) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 microg ml(-1)), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 microg ml(-1)). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Identification of GFAT1-L, a novel splice variant of human glutamine: fructose-6-phosphate amidotransferase (GFAT1) that is expressed abundantly in skeletal muscle.

Glutamine:fructose-6-phosphate amidotransferase (GFAT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway, which plays an important role in hyperglycemia-induced insulin resistance. To evaluate the role of GFAT1 expression, we analyzed the expression profiles of GFAT1 mRNA in various human tissues using reverse transcriptase-polymerase chain reaction. We report here the identification and cDNA cloning of a novel GFAT1 splice variant expressed abundantly in skeletal muscle and heart. This subtype, designated GFAT1-L, contains a 54-bp insertion within the GFAT1 coding sequence. Recombinant GFAT1-L protein possessed functional GFAT activities and biochemical characteristics similar to GFAT1. Previously, GFAT1 was considered a simplex enzyme. The identification of a novel GFAT1 subtype possessing functional enzymatic activity and tissue-specific expression should provide additional insight into the mechanism of skeletal muscle insulin resistance and diabetes complications.

Neuropeptide Y in central control of feeding and interactions with orexin and leptin.

Neuropeptide (NPY) increases feeding when injected into the brain. In this study, we tested the hypothesis that its action might be related to feeding regulation of the orexin and leptin systems in rats. Intracerebroventricular administration of NPY (1 nmol/5 microL) stimulated feeding in rats. Injection of an antibody to orexin-A inhibited feeding, suggesting that endogenous orexin exerts a stimulatory tone on feeding. Intracerebroventricular injection of orexin antiserum before injection of NPY significantly attenuated the feeding response to NPY. On the other hand, ip pretreatment with leptin (2 mg/kg) significantly decreased food intake and inhibited NPY-induced feeding. We then examined whether orexin-containing neurons are activated under the stimulation of feeding in response to intracerebroventricular NPY or suppression of feeding in response to ip leptin, using Fos-like immunoreactivity (FLI) as a marker of neural activation. We observed that FLI was induced in the paraventricular, supraoptic, and dorsomedial nuclei as well as the lateral hypothalamic area (LHA) following administration of NPY. Double staining with anti-Fos and antiorexin antibodies revealed that 23.4% of the orexin-containing neurons in the LHA expressed FLI after NPY injection. Approximately 7.8% of the orexin-positive neurons in the LHA coexpressed Fos after leptin plus NPY. Our data indicate that a functional interaction among NPY, orexin, and leptin exists that may contribute to the central regulation of appetite.

Central administration of neuromedin U activates neurons in ventrobasal hypothalamus and brainstem.

Neuromedin U (NMU) is a peptide isolated from the porcine spinal cord. Recently, two receptors for NMU have been identified and characterized. A recent study indicated that NMU is an anorectic chemical in the brain. The present study shows that NMU has an action in the brain to inhibit food intake in rats. Intracerebroventricular injection of NMU inhibited dark-phase feeding. Animals injected with NMU showed a strong increase in Fos-immunoreactive nuclei in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, and in the parabrachial nucleus of the brain stem. Double immunohistochemistry revealed that a high number of oxytocin-immunoreactive neurons in the PVN and SON contained Fos after intracerebroventricular injection of NMU. In addition, a small proportion of vasopressinergic cells within the PVN and SON were found to contain Fos. The effect of NMU on the hypothalamus and brain stem contributes to the inhibitory effects of NMU on feeding behavior.

Effects of central and peripheral injection of leptin on food intake and on brain Fos expression in the Otsuka Long-Evans Tokushima Fatty rat with hyperleptinaemia.

Hyperleptinaemia is observed in obese animals and humans, suggesting that leptin resistance rather than leptin deficiency is a characteristic feature of obesity. This study was designed to determine whether peripherally or centrally administered leptin is effective on the short-term food intake and expression of Fos protein in the hypothalamus in the Otsuka Long-Evans Tokushima Fatty (OLETF) or Long-Evans Tokushima Otsuka (LETO) rat, as a control. The OLETF rat exhibits a polygenic syndrome of hyperphagia, obesity, hyperinsulinaemia, and hyperglycaemia. Male OLETF rats of 5, 8, and 14 weeks of age became heavier than LETO rats. Serum leptin concentrations were not significantly different between LETO and OLETF rats at the age of 5 weeks, but in 8- and 14-week-old OLETF rats were increased to 3.4 and 2.9 times those of LETO rats, respectively. The 8-week-old OLETF and LETO rats were given intraperitoneal (i.p.) injections with recombinant mouse leptin to measure the kinetics. There was a dramatic increase in plasma leptin concentration at 1 h, a decline by 3 h, and the concentrations 6 h after injection were similar to the basal levels. There were no significant difference between OLETF and LETO rats. In LETO rats at 5, 8 and 14 weeks of age, i.p. injection of leptin significantly decreased food intake. Whereas 5-week-old OLETF rats responded to leptin with a decrease in food intake, 8- and 14-week-old OLETF rats became resistant to peripherally administered leptin. In contrast, intracerebroventricular (i.c.v.) injections of leptin were very effective in inhibiting food intake in both OLETF and LETO rats at 14 weeks of age. Intraperitoneal injection of leptin in the LETO rats at each age increased the number of Fos-positive nuclei detected in the ventromedial hypothalamic (VMH), the dorsomedial hypothalamic (DMH) and arcuate nuclei, whereas there was no significant increase in the number of cells expressing c-fos protein in the hypothalamus of the 8- and 14 week-old OLETF rats with hyperleptinaemia. On the other hand, increased expression of c-fos protein in the VMH, DMH and arcuate nuclei following i.c.v. injection of leptin was observed in both OLETF and LETO rats at 5, 8 and 14 weeks of age. These data demonstrated that obese OLETF rats are peripherally leptin resistant, while they retain sensitivity to centrally administered leptin.

Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues.

We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Immunosuppressant agent FK506 stimulates growth hormone (GH) secretion and gene expression of hypothalamic GH-releasing hormone in the rat.

Immunosuppressant agent FK506 has been reported to stimulate ACTH release from pituitary cells. We examined the effects of FK506 on GH release from the rat anterior pituitary cells and the effects of FK506 on hypothalamic GH- releasing hormone (GRH) and somatostatin (SS) gene expression in conscious male rats. In vitro experiments, the monolayer pituitary culture and reverse hemolytic plaque assay were employed to examine the GH release from the rat anterior pituitary cells. In in vivo experiments, the FK506 was administered for 7 days and then sequential blood sampling was performed every 20 min during 6 h in conscious rats. The hypothalamus was removed, and total RNA was extracted for Northern blot analysis. The FK506 significantly stimulated GH release from the rat anterior pituitary cells in a dose-dependent manner in vitro. In in vivo experiments, the area under the curve of GH surges was significantly increased in FK506-treated rats, although the peak height and the trough level of GH surges were not altered. Pituitary GH messenger RNA (mRNA) levels were significantly increased by the FK506 treatment. Hypothalamic GRH mRNA levels were significantly increased in FK506- treated rats, whereas hypothalamic SS mRNA levels were not altered. These findings indicate that FK506 stimulates GH secretion and gene expression of hypothalamic GRH in the rat.

Induction of Fos protein in the rat hypothalamus elicited by insulin-induced hypoglycemia.

To evaluate the responses to insulin-induced hypoglycemia of neurons in vivo, we studied Fos protein induction in the brain by means of immunohistochemistry. The induction of Fos protein was maximum after the first injection of insulin for 3 h. This induction was found in the parvocellular division of paraventricular nucleus (PVN), the periventricular, dorsomedial and arcuate nuclei and the lateral hypothalamic area of the hypothalamus. These findings show the activation of specific subsets of neurons in areas of the hypothalamus following hypoglycemic stimulation.

Central glucopenia induced by 2-deoxy-D-glucose stimulates somatostatin secretion in the rat.

The mechanisms involved in 2-deoxy-D-glucose (2-DG)-induced growth hormone (GH) suppression in the rat were examined. Conscious male rats were given 2-DG by intracerebroventricular (icv) injection and the pulsatile GH secretion was monitored for 6 h. The single icv injection of 2-DG (8 mg/rat) eliminated pulsatile GH secretion in conscious rats. Pretreatment with somatostatin (SS) antiserum completely restored the suppressed GH secretion in the 2-DG treated rats. Hypothalamic GH-releasing hormone (GRH) and SS mRNA levels were not altered by single and multiple icv injections of 2-DG. These findings suggest that 2-DG-induced GH suppression is primarily due to hypersecretion of SS without a significant change at the transcription level in the rat.

Inhibitory effects of interleukin-1 on growth hormone secretion in conscious male rats.

Although various pathophysiological effects of interleukin (IL) on the CRF-ACTH-adrenal axis and gonadotropin secretion have been studied extensively, the effects of IL on GH secretion still remain to be elucidated. We investigated the possible effects of IL on GH secretion in six groups of conscious rats. In four groups, IL was administered by continuous iv infusion and in the other two, by intracerebroventricular injection. Saline-treated rats served as controls for these groups. Sequential blood sampling was performed every 20 min in all groups, and the plasma GH concentration was determined by RIA. The expression of hypothalamic c-fos protein in a separate group was examined by immunohistochemistry. Continuous infusion of both IL-1 alpha and IL-1 beta (10 ng/min) significantly inhibited GH surges. The plasma IL-1 level was elevated to 2-3 ng/ml. Continuous iv infusion of IL-2 and IL-6 had no effect on GH secretion. The intracerebroventricular injection of both IL-1 alpha and IL-1 beta significantly inhibited GH surges, and the inhibitory effect was much greater for IL-1 beta than for IL-1 alpha. Continuous iv infusion of IL-1 beta markedly stimulated c-fos expression in specific hypothalamic nuclei, particularly in the paraventricular nucleus. These findings suggest that, in the rat, IL-1 inhibits GH secretion through its peripheral and central actions.

Differential gene expression of growth hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues.

Growth hormone (GH)-releasing hormone (GRH) acts on specific receptors in the anterior pituitary to stimulate the synthesis and release of GH. Recent reports suggest that GRH is also synthesized in extrahypothalamic tissues. To evaluate the potential roles of extrahypothalamic GRH, we studied the gene expression of GRH and GRH receptors in various rat tissues by reverse transcribed (RT)-polymerase chain reaction (PCR). Total RNA was extracted from twenty-three rat organs and RT-PCR was performed with GRH and GRH receptor primers. Highly-sensitive RT-PCR-Southern blotting showed that GRH and GRH receptor mRNA coexist in the widespread tissues (14 of 25 tissues). GRH mRNA was relatively abundant in the cerebral cortex, brain stem, testis, and placenta, while GRH receptor mRNA was abundant in renal medulla and renal pelvis. Northern blot hybridization using poly A+ RNA indicated that the transcript of GRH receptor gene found in the renal medulla was similar to the longer transcript (about 4 Kb) of pituitary GRH receptor in the size. These results suggest that GRH plays a potential role not only in the neuroendocrine axis, but also in the autocrine and paracrine systems in extrahypothalamic tissues.

Gene expression of hypothalamic growth hormone (GH)-releasing hormone and somatostatin does not correlate with pulsatile secretion of GH in the adult mouse.

The secretion of growth hormone (GH) shows a pulsatile pattern in many mammalian species, and depends on the interaction of two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIH). A surge of GHRH secretion into the hypophysial portal blood induces peak secretion of GH from the anterior pituitary. To study whether the rhythmic and reciprocal oscillation in secretion of GHRH and SRIH are associated with changes in synthesis of these peptides, we examined the expression of hypothalamic GHRH and SRIH mRNA by Northern blotting during trough and peak phases of GH secretion in male mice. Hypothalamic GHRH and SRIH mRNA levels did not differ between trough and peak phases of GH secretion. This result suggests that changes in GHRH and SRIH secretion have little association with changes in the synthesis of these peptides at the hypothalamus in the male mouse.