Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression.

Despite its beneficial role in host defense mechanisms, excessive nitric oxide (NO) production by activated macrophages has been implicated in several inflammatory diseases. To clarify the mechanisms of anti-inflammatory activities of Polygonum tinctorium, we evaluated whether extracts of P. tinctorium could modulate the production of NO by activated macrophages. An AcOEt extract of P. tinctorium markedly inhibited NO synthesis by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages and the macrophage-like cell line RAW 264.7 in a dose-dependent manner. Inhibition of NO synthesis was achieved by reducing inducible NO synthase (iNOS) expression at protein and mRNA levels. However, the AcOEt extract of P. tinctorium failed to inhibit NO synthesis when iNOS was already expressed following stimulation with IFN-gamma and LPS. The AcOEt extract also exhibited inhibitory activity on iNOS expression in human lung epithelial A549 cells stimulated with a combination of IFN-gamma, TNF-alpha and IL-1 beta without affecting the expression of constitutive isoforms of NOS. Furthermore, in vivo injection of the AcOEt extract of P. tinctorium into LPS-treated mice significantly reduced NO synthesis by peritoneal exudate cells under ex vivo conditions. These results suggest that P. tinctorium extract may be a potential therapeutic modulator of NO synthesis in various pathological conditions.

[Inhibition of antigen-specific T helper type 2 responses by Perilla frutescens extract].

Perilla frutescens leaf extract (PFE) is known as a natural medicine with anti-allergic activities, although its mechanism of action remains unclear. In this study, we examined the effect of PFE on antigen-specific antibody and on cytokine production. Mice were immunized three times (weekly) with sugi basic protein (SBP), a major allergen of Japanese cedar pollen, in alum adjuvant. PFE was injected intraperitoneally into mice on day 2 before and on the day of each immunization with SBP in alum adjuvant. Serum anti-SBP IgE and IgG 1 antibody levels were significantly suppressed in mice injected with PFE. Furthermore, the production of interleukin (IL)-4, IL-5 and IL-10 by SBP-stimulated splenocyses also decreased in PFE-injected mice in a dose-dependent manner. However, PFE had no effect on either the serum anti-SBP IgG 2 a antibody levels or on interferon (INF)-gamma production by splenocytes. When splenocytes were stimulated with concanavalin A, there was no difference in cytokine production between mice injected with PFE and control mice injected with vehicle. SBP-specific T cell line established in the presence of PFE from the lymph node cells of mice immunized with SBP showed reduced IL-4, IL-5 and IL-10 production compared with that established in the absence of PFE. In contrast, comparable levels of IFN-gamma production were observed between these two T cell lines. These data suggest that PFE down-regulates Th 2-type cytokine production and prevents the Th 1/Th 2 balance from polarizing toward Th 2-type immune responses.

Regulation of plasma and liver total cholesterol levels by dietary oleic acid in rats fed a high-cholesterol diet.

The effects of diets containing fats and oils or fatty acids on the lipid metabolism were investigated in male rats of the Wistar strain fed hypercholesterolemic diets, especially focusing our attention on the correlation between dietary oleic acid (OLE) contents and the levels of plasma and liver total cholesterol (T-CHOL) or the fatty acid profiles in plasma and liver CHOL-ester. In the rats fed the free (FR)-type fatty acids, the concentrations of plasma and liver T-CHOL were high and the amounts of neutral steroids excreted into the feces were low when compared with those of rats given the triacylglycerol (TG)-type fatty acids, showing that TG-type fatty acids suppress the intestinal CHOL absorption more than the FR-type fatty acids do. The concentrations of plasma T-CHOL were highest in rats fed the oleic acid (OLE)-rich diets, followed in order by rats supplied with the palmitic acid (PAL)-rich diets, the hydrogenated coconut oil (HCO) diet, and the linoleic acid (LIN)-rich diets; the lowest was in rats given tristearin (TSTE) and linseed oil (LIS) diets. A positive correlation was obtained between the dietary OLE contents and the levels of plasma and liver T-CHOL or OLE in the plasma and liver CHOL-ester, and an inverse correlation between dietary OLE contents and the amounts of excreted neutral steroids. These results suggest that the dietary OLE contents regulate the levels of plasma and liver T-CHOL in CHOL-loaded rats.

Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats. II. Role of enhancement of fructose metabolism and overproduction of triglyceride in the liver and kidney on hepatorenal toxicity of acetaminophen.

Fructose-induced hypertriglyceridemic rats are resistant to hepatoxicity and susceptible to nephrotoxicity of acetaminophen (APAP) as compared with normal ones. The present studied were designed to evaluate how fructose-treatment affects the developmental mode of hepatorenal toxicity of APAP. First, following fructose-pretreatment for various durations (1 day, 1 week or 3 weeks), 1-day-fructose-pretreatment induced hypertriglyceridemia and enhancement of APAP-nephrectoxicity simultaneously. However, it took at least 3 weeks for fructose-pretreatment to reduce APAP-hepatotoxicity. Second, following fructose, sucrose or glucose-pretreatment for 3 weeks, fructose-pretreated rats showed marked hypertriglyceridemia and modification of APAP-hepatorenal toxicity. Sucrose-pretreated rats showed less effects than fructose-pretreated rats. Glucose-pretreated rats showed no changes in plasma triglyceride and APAP-hepatorenal toxicity. Third, rats with hypertriglyceridemia induced by olive oil or Triton WR-1339 which did not produce enhanced metabolism and triglyceride-overproduction in the liver and kidney showed no modification of APAP-hepatorenal toxicity. Pretreatment of glycerol which was metabolized in liver and kidney and induced an overproduction of triglyceride resulted in an enhancement of APAP-nephrotoxicity. These results indicate that an enhancement of fructose metabolism and an overproduction of triglyceride in liver and kidney are responsible for the modification of APAP-hepatorenal toxicity in fructose-induced hypertriglyceridemic rats.

Intraventricular administration of estradiol modulates rat prolactin secretion and synthesis.

The effect of estradiol (E2) on rat tuberoinfundibular dopaminergic (TIDA) neurons was examined in vivo, employing chronic intraventricular (i.c.v.) infusion technique using an osmotic mini-pump. The activity of TIDA neurons was assessed by the release and synthesis of prolactin (PRL) in the rat pituitary gland and by the changes in the 3, 4-dihydroxyphenylacetic acid (DOPAC) and dopamine (DA) levels and in the DOPAC/DA ratio in the rat hypothalamus. We also examined the [3H] E2 binding in the rat hypothalamus. Ovariectomized female Wistar rats with E2 replacement were treated with daily i.c.v. infusion of 1 microM of E2 or saline vehicle for 1, 3, and 7 days using the Alzet osmotic mini-pump and brain infusion kit. At 1 day of i.c.v. infusion of E2, the serum PRL level was significantly decreased compared with that in the vehicle group. Northern blot analysis of the total RNA isolated from the pituitary glands demonstrated a decrease in the PRL gene transcript level in the E2 group. At 3 days of E2 treatment, however, the serum PRL level was significantly increased compared with that of the vehicle-injected group and Northern blot analysis also demonstrated that the PRL gene transcript level was increased in the E2 group. At 7 days of E2 administration, there were no significant differences between the E2 and vehicle groups in either serum PRL or PRL gene transcript levels. There was a significant increase in the DOPAC/DA ratio after 1 day in the E2 group. However, no significant effects of E2 on this ratio were observed at 3 and 7 days of treatment. The DOPAC concentration in the E2 group was significantly increased at day 1 and significantly decreased at day 3, compared with that of the respective time in vehicle group. At day 7 there was no significant change in DOPAC concentration in either groups. The DA concentrations in the hypothalamus was not changed on any day in either group. Specific [3H] E2 binding was observed in the rat hypothalamus. These data suggest that E2 may have a biphasic effect on the accumulation of PRL gene transcripts and on the PRL secretion in the rat pituitary by first stimulating and then inhibiting the TIDA neuronal activity.

Effects of sensory stimulation on respiratory cardiac cycle variability in humans.

The effects of the phase of respiration on the response of respiratory cardiac cycle variability to sensory stimulation were studied in five healthy young male subjects. Transcutaneous electrical stimulation of the ulnar nerve or hand-grip exercise was applied during inspiration or expiration. Although both electrical stimulation and hand-grip exercise depressed respiratory cardiac cycle variability, the nature of the depression differed according to where in the respiration cycle the stimuli were applied. The amplitude of respiratory cardiac cycle variation was significantly decreased when either stimulus was applied during expiration (P < 0.05), and was unchanged when applied during inspiration (P > 0.05). These findings would suggest that cardiac vagal efferent activity was effectively inhibited by sensory stimulation during expiration, but was not inhibited by such stimulation during inspiration. This mechanism may account, in part, for the known suppression of respiratory cardiac cycle variability during exercise.

[Enzyme-linked immunosorbent assay for the quantification of Cry j I and Cry j II].

Enzyme-linked immunosorbent assays (ELISA) were developed to specifically quantify the two major allergens from Japanese cedar pollen, Cry j I and Cry j II. Polystyrene microplates coated with antibodies specific for Cry j I or Cry j II were incubated with an allergen and then with biotinylated anti-Cry j I or Cry j II antibody. The bound allergen-biotin Ab complexes were detected with HRPO-conjugated streptavidin and an enzyme substrate. The working ranges of Cry j I ELISA and Cry j II ELISA were 0.3-20 ng/ml and 0.6-20 ng/ml, respectively. Intra- and inter-assay coefficients of variation for reproducibility were 1.5-10.3% and 0.9-12.9%. These ELISA systems showed no cross-reactivity between Cry j I and Cry j II and showed little cross-reactivity with pollen allergens of plants botanically related to the Japanese cedar. Using the Cry j I ELISA and the Cry j II ELISA, it was possible to quantify Cry j I and Cry j II easily and accurately. These ELISA systems will be useful in various fields, especially for the analysis and standardization of the allergens necessary for diagnosis and treatment of Japanese cedar pollinosis.

Prolactin stimulates [3H]dopamine release from dispersed rat tubero-infundibular dopaminergic neurons and dopamine decreases gonadotropin-releasing hormone release induced by calcium ionophore.

In order to investigate the involvement of prolactin-dopamine and dopamine-gonadotropin interactions in the hypothalamo-pituitary axis of hyperprolactinemia, in vitro studies were performed using primary cultures of dispersed rat hypothalamic heterogeneous cells containing tubero-infundibular dopaminergic neurons or gonadotropin-releasing hormone (GnRH) neurons. We observed that prolactin caused dose-dependent stimulation of [3H]dopamine release after a 16-h incubation. Staurosporin (10 nmol/l), an inhibitor of protein kinase C, significantly reduced the [3H]dopamine release induced by prolactin (1 mg/l). Incubation of tubero-infundibular dopaminergic neurons with prolactin (1 mg/l) had no effect on intracellular cyclic adenosine monophosphate accumulation. Dopamine (1 mumol/l) significantly (p < 0.01) reduced the release of GnRH induced by 50 mumol/l calcium ionophore from dispersed hypothalamic cells from the preoptic area, while prolactin had no effect on GnRH release. These data support the hypothesis that the antigonadotropic effect of prolactin on the hypothalamus is mediated by an inhibitory effect of dopamine on GnRH release.

Intraventricular administration of histidyl-proline-diketopiperazine [Cyclo(His-Pro)] suppresses prolactin secretion and synthesis: a possible role of Cyclo(His-Pro) as dopamine uptake blocker in rat hypothalamus.

Histidyl-proline-diketopiperazine [Cyclo (His-Pro) (CHP)] was discovered to be one of the metabolites of TRH. To understand the specific role of CHP in rat hypothalamic dopamine neurons, we examined the in vivo effects of intraventricular (icv) infusion of CHP on the release and synthesis of PRL in the rat pituitary and the 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine ratio in the rat hypothalamus. We also examined the in vitro effects of CHP on the release of [3H]dopamine from dispersed tuberoinfundibular dopamine neurons, [3H]dopamine reuptake in hypothalamic membrane fractions, and PRL release from rat pituitary cultured cells. Female rats were treated by icv infusion of 1 microM CHP daily for 1, 3, and 7 days, using Alzet osmotic pumps. After 1 day of treatment, the serum PRL concentration was significantly decreased. Northern blot analysis of the total RNA isolated from the pituitary glands of control animals using 32P-labeled PRL cDNA as a probe indicated the presence of PRL gene transcript, 1.0 kilobase in size, and its amount was decreased by CHP treatment. CHP did not affect [3H]dopamine release from dispersed tuberoinfundibular dopaminergic neurons at any concentration up to 1 microM. CHP did not inhibit PRL release from cultured pituitary cells at low concentrations (1-100 nM), but it stimulated PRL release at high concentrations (1 and 10 microM). We also examined the concentrations of dopamine and DOPAC in the rat hypothalamus when CHP was administered icv for 1 or 7 days. There was a significant decrease in the DOPAC/dopamine ratio after CHP treatment for 1 day. Furthermore, CHP caused dose-dependent inhibition of [3H]dopamine uptake by the rat hypothalamus similar to other dopamine uptake blockers, such as benztropine and GBR12909. These data suggest that icv administration of CHP might decrease both PRL secretion and accumulation of PRL gene transcripts in the pituitary by decreasing the DOPAC/dopamine ratio and inhibiting dopamine reuptake in the rat hypothalamus.

Intraventricular administration of thyrotrophin-releasing hormone (TRH) suppresses prolactin secretion and synthesis: a possible involvement of dopamine release by TRH from rat hypothalamus.

The administration of thyrotrophin-releasing hormone (TRH) causes a variety of dopamine-related biological events. To understand the specific role of TRH on rat hypothalamic dopamine neurones, we examined the in-vivo effects of intraventricular (i.c.v.) infusion of TRH on the release and synthesis of prolactin in the rat pituitary gland and on the changes in binding of [3H]MeTRH and dopamine turnover rates in rat hypothalamus. We have also examined the in-vitro effects of TRH on the release of [3H]dopamine from dispersed tuberoinfundibular dopamine neurones. Female rats were treated with i.c.v. infusions of 1 mumol TRH/1 daily for 1, 3 and 7 days using Alzet osmotic pumps. Following 7 days of treatment the serum prolactin concentrations were significantly decreased. A reduction in hypothalamic TRH-binding sites (Bmax) was also apparent but the dissociation constant (Kd) was unaffected. Northern blot analysis of total RNA isolated from the pituitary glands of control animals using 32P-labelled prolactin cDNA as a probe indicated the presence of three species of prolactin gene transcripts of approximately 3.7, 2.0 and 1.0 kb in size, and these were decreased by TRH treatment. We examined the turnover rate of dopamine in the rat hypothalamus when TRH was administered i.c.v. for 7 days. There was a significant increase in 3,4-dihydroxyphenylacetic acid/dopamine ratio with TRH treatment. Moreover, exposure to TRH stimulated [3H]dopamine release from rat tuberoinfundibular neurones in a time- and dose-dependent manner. Dopamine receptor antagonists such as SCH23390 and (-)sulpiride, and other neuropeptides such as vasoactive intestinal peptide and oxytocin did not affect TRH-stimulated [3H]dopamine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of TNF-alpha on prolactin secretion from rat anterior pituitary and dopamine release from the hypothalamus: comparison with the effect of interleukin-1 beta.

The effects of human recombinant interleukin-1 beta and -6 and tumor necrosis factor-alpha (TNF-alpha) on the releases of PRL and dopamine were examined using monolayer cultures of rat pituitary cells and hypothalamic cells. The release of PRL from rat pituitary cells in 30 min was increased about 2-fold (p less than 0.05) by 10(5) U/l interleukin-1 beta, 10(5) U/l interleukin-6 or 100 micrograms/l TNF-alpha. TNF-alpha at 100 micrograms/l significantly increased PRL release within 5 min incubation and this effect continued throughout the next 30 min of incubation. Incubation for 5 min with TNF-alpha caused dose-dependent stimulation of PRL release. These cytokines did not modulate [3H]-dopamine release from primary cultures of hypothalamic cells. These results suggest that these cytokines stimulate PRL release directly at the pituitary gland, without modifying the release of dopamine from the hypothalamus.

4-Hydroxytamoxifen binds to estrogen receptors and inhibits the growth of human endometrial cancer cells in vitro.

Effects of 4-hydroxytamoxifen, a major metabolite of tamoxifen, on the proliferation of cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in primary culture. Competitive binding studies showed that 4-hydroxytamoxifen effectively binds to cytoplasmic estrogen receptors (ER) in uterine adenocarcinomas. Of 20 endometrial adenocarcinomas examined, five tumors were successfully grown in primary cell culture. The addition of 4-hydroxytamoxifen (1 nmol/l to 1 mumol/l) in a medium supplemented with estrogen-free serum resulted in a dose-dependent inhibition of the growth of cancer cells in two tumors having ER. However, 4-hydroxytamoxifen did not affect the growth in the culture system of the remaining three tumors, in which ER were absent in two tumors but were present in one. These results strongly suggest that tamoxifen has a direct growth-inhibitory effect on human endometrial adenocarcinoma possibly through ER in the tumor.

Primary hypothyroidism in an adult patient with protein-calorie malnutrition: a study of its mechanism and the effect of amino acid deficiency.

A man with diabetes mellitus, chronic hepatitis, chronic pancreatitis, and blind loop syndrome but without any previous thyroid disease developed three episodes of transient primary hypothyroidism associated with protein-calorie malnutrition (PCM). Clinical examinations suggested that this primary hypothyroidism was not caused by chronic thyroiditis, iodine deficiency, or iodine excess. Since the three times association of primary hypothyroidism with PCM suggested the possibility that the primary hypothyroidism was caused by PCM, we have tried to clarify its mechanism. For this purpose we have investigated the change of thyroid functions during protein-calorie repletion and the effect of amino acid deficiency. Total parenteral nutrition with full supplementation of amino acids resulted in a rapid increase in serum thyroxine (T4), triiodothyronine (T3), free T4, and reverse T3, and subsequently, a rapid decrease in TSH in several days after the nutrition was begun. When amino acid solution was changed to that depleted of phenylalanine and tyrosine after the restoration of thyroid functions, serum T4 and T3 showed a gradual decrease, but serum free T4 and TSH remained within normal range. However, resupplementation of phenylalanine and tyrosine after 8 weeks of depletion gave a rapid increase in serum T4, T3, free T4, and reverse T3. These results suggested that the primary hypothyroidism was caused by an impaired T4 production and that the deficiency of amino acids in PCM partly contributed to the impairment of T4 production.