Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)     

介绍一种LHRH调节LH分泌的离体模型—分散垂体细胞灌流技术

成年雄性 SD 大鼠断头后分离出垂体前叶(anterior pituitary,AP)。用胰蛋白酶消化和机械分散制备 AP 细胞(成活率大于95%)。分散的细胞悬液与生物凝胶混合后装上灌流柱,然后用 M199溶液连续灌流24h 以上。每间隔1~h 给予一次6min 的 LHRH 脉冲式刺激。细胞在此灌流过程中有一个稳定的基础 LH 分泌水平。LHRH 刺激能迅速引起 LH 分泌。对同一剂量 LHRH 的多次刺激可产生相同的 LH 脉冲。在一定的 LHRH 浓度范围内(1×10~(-10)_1×10~(-7)mol/L),LH 分泌与 LHRH 的剂量-效应曲线呈线性。实验结果表明,连续灌流分散的 AP 细胞的技术,优于单层细胞培养和组织块灌流等其他方法,是一种较为理想的研究LHRH 调节 LH 分泌机理的体外模型。     

Effects of dopamine and norepinephrine on in vitro spontaneous and gonadotropin-releasing hormone-induced gonadotropin release by dispersed cells or fragments of the goldfish pituitary

Enzymatically dispersed goldfish pituitary cells or freshly prepared goldfish pituitary fragments continue to secrete gonadotropin spontaneously in a column perifusion system. After the establishment of basal secretion rates, treatment of dispersed pituitary cells with 5 and 500 nM dopamine, or pituitary fragments with 50 and 100 nM dopamine, decreased the amount of gonadotropin released into the perifusate. Perifusion with 500 nM dopamine also abolished the gonadotropin-release response to a 10 nM solution of a luteinizing hormone-releasing hormone analogue in both perifusion systems. Perifusion of pituitary dispersed cells or fragment preparations obtained from sexually regressed goldfish with 50 nM norepinephrine consistently increased the amount of gonadotropin released into the perifusate. These results provide in vitro evidence for direct dopamine inhibition of spontaneous gonadotropin release, blockade by dopamine of gonadotropin-releasing hormone actions, and norepinephrine stimulation of gonadotropin secretion in goldfish.     

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Dialyzability and binding of digoxin-like immunoreactive factors (DLIF) with serum macromolecules in uremic patients on hemodialysis

Digoxin-Like Immunoreactive Factors (DLIF) which cross-react with antidigoxin antibodies are present in elevated concentrations in patients on hemodialysis, uremia, hypertensives, liver failure, pre-eclampsia and premature birth. DLIF may have a potential role as a natriuretic hormone with a speculated low molecular weight (less than 1000). We studied the dialyzability and bindings of DLIF with serum components in hemodialysis patients. We analyzed DLIF concentrations in sera and protein free ultrafiltrates of 31 patients and 22 normal volunteers using a fluorescence polarization assay for digoxin. The DLIF concentrations were expressed as nmol/L Digoxin Equivalent. The gel filtration analysis was done using three different Bio-Gel columns with molecular weight cut-offs of 10,000, 20,000 and 40,000. Molecules with lower molecular weight than cut-off were absorbed in the column. Only 3 out of 22 normal volunteers (13.6%) showed measurable DLIF. However 23 out of 31 patients (74.2%) showed measurable DLIF. The concentrations of DLIF were significantly higher in patients with renal failure on hemodialysis (P less than 0.05) by both chi-squared and Fisher's exact test. We observed no statistically significant difference in the concentrations of DLIF in pre and post-dialysis sera, indicating that DLIF were not filtered during hemodialysis. We observed no DLIF activity in the protein free ultrafiltrates of any DLIF positive sera (patients and normal volunteers), indicating that unlike digoxin (where we observed 70-80% of total digoxin concentrations in ultrafiltrates), DLIF were strongly bound to serum components. With Bio-Gel filtration experiments (five different serum pools), we recovered all DLIF activities in the fraction equivalent to the void volume of the column with Bio-Gel P6 and P10 columns, indicating that DLIF were almost completely bound to serum components with molecular weight greater than 20,000. On the other hand, we recovered no DLIF activities in the void volume when the same serum pools were passed through the Bio-Gel P30 column, indicating that DLIF were strongly bound to serum macromolecules with molecular weight less than 40,000. In sharp contrast, when serum containing digoxin was subjected to the same series of experiments, we recovered only 20-30% of digoxin concentrations in void volume with all three columns as expected since digoxin is only 25% bound to albumin (MW 67,000).     

Long-term maintenance of growth hormone secretion in perifused rat anterior pituitary cells

We studied the effect of rat growth hormone-releasing factor-(1-43) acid, rGRF(1-43)OH, on the long-term secretion of rat growth hormone (rGH) in dispersed primary cultured cells of rat anterior pituitaries over a period of 7 days or longer. Results of the perifusion assay show that freshly dispersed cells secrete more rGH than 4-day-old redispersed cells (P less than 0.05), that a stabilization period ranging from 4 to 24 h allows a greater production of rGH per day than longer periods (P less than 0.05) and that the working concentrations of rGRF-(1-43)OH and prostaglandin E2 (PGE2) that insured the best responsiveness and longer viability are 50 pM and 10-1000 nM, respectively. Under these conditions, the cells continued secreting rGH after 42 days of perifusion, and 315 milligrams of rGH was produced over that period.     

Steroidogenic effect of 17 beta-estradiol on rabbit luteal cells in vitro: estrogen-induced maintenance of progesterone production

Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production.     

Effects of gonadal steroids on follicle-stimulating hormone and luteinizing hormone secretion by pituitary cells from castrated and intact male rats

The effectiveness of androgens in suppressing gonadotropin secretion declines with time following orchidectomy; however, the mechanism for this acquired resistance to androgen action is unknown. The role of the pituitary was studied by use of perifused rat pituitary cells and cells in monolayer culture. Pituitary cells from 7-wk-old intact male rats and rats that had been castrated 2 wk previously were treated with 10 nM testosterone (T) for 24 h; cells were then packed into perifusion chambers and stimulated with 2.5 nM GnRH for 2 min every hour for 8 h during which time T treatment was continued. T suppressed GnRH-stimulated LH secretion and LH pulse amplitude equally in both groups to approximately 60% of control values. Interpulse LH secretion was unchanged by T in either group. GnRH-stimulated FSH release was suppressed more (p less than 0.05) by T with cells from castrated rats than with cells from intact rats (76 +/- 4% vs. 90 +/- 2% of control; mean +/- SEM). By contrast, the action of T to increase interpulse basal FSH secretion was less (p less than 0.05) with cells from castrated rats (115 +/- 10% of control) than with cells from intact rats (146 +/- 6% of control). T treatment for 72 h also increased basal FSH secretion by pituitary cells in monolayer culture to a lesser extent with cells from castrated rats than with cells from intact rats (151 +/- 14% vs. 191 +/- 16% of control, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin pretreatment facilitates GRF-induced GH release and increase in free calcium in pituitary cells

Somatostatin pretreatment sensitizes rat anterior pituitary to hGRF stimulation in vitro. The pretreatment (1 nM for 10 min) facilitated GH release response of dispersed rat anterior pituitary cells to hGRF (1 nM for 3 min) 2.04-fold in a perifusion system. The effect lasted even 20 min after the pretreatment. SRIF pretreatment decreased cAMP content in the cells after hGRF stimulation to 61% of the control value. When hGRF was replaced by 1 mM DBcAMP and 15 mM KCl, the pretreatment increased GH secretion 1.69- and 1.67-fold respectively. SRIF pretreatment (1 nM for 10 min) caused a larger increase in (Ca2+)i by hGRF than that of control. The effect of SRIF pretreatment facilitates GRF-induced increase in GH secretion probably through the stimulation of increase in (Ca2+)i.     

In vitro secretion of somatostatin (SRIH) by human adenomatous somatotropic cells. Relation with somatotropic hormone (GH) release and modulation by thyroliberin (TRH)

SRIH and GH secretions by GH-secreting adenomatous human pituitary cells were analyzed in vitro in a perifusion system. Of the 13 adenomas studied, 7 secreted SRIH, in variable amounts (50 to 700 pg/ml/2 min., corresponding to 600 10,700 pg for the total experiment. SRIH secretion increased during the perifusion, the highest levels being observed at the end of the perifusion. GH secretion also varied from one adenoma to the other (6 to 500 ng/ml). In most cases, the secretion profiles were negatively correlated, GH secretion decreasing while SRIH secretion was increasing. In the presence of 10(-7) M TRH, GH secretion increased while that of SRIH decreased. The hypothesis of a paracrine and/or an autocrine role for SRIH as well as its possible in situ synthesis are discussed.     

Highly efficient genetic transformation of Bacillus subtilis attached to sand grains

Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 degrees C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml-1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 microgram ml-1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Effect of naloxone and beta-casomorphin on the hypothalamic-pituitary-luteinizing hormone axis in vitro.

The effect of naloxone and beta-casomorphin on luteinizing hormone (LH) release from pituitary cell aggregates, obtained by three-dimensional culture, with or without mediobasal hypothalamic fragments was studied in vitro. Short-term naloxone perifusion at a concentration of 10(-5)M did not modify either basal or LHRH-stimulated LH release from the pituitary cell aggregates. In contrast, a 12-min naloxone perifusion at the same concentration caused an increase in LH release in the mediobasal hypothalamic-pituitary cell aggregate axis. This increase was rapid (12-16 min after time pulse), marked [up to 10 times (p less than 0.004) the initial base line], short (return to the base line secretion 32-40 min after the beginning of the time pulse) and dose-dependent, with a rise greater than 1000% at a concentration of 10(-4) (p less than 0.006). The same effect was observed when a second pulse was applied 48 min after the first one. LH release induced by naloxone was antagonized 56 +/- 2% (p less than 0.03) by beta-casomorphin (an exogenous opiate) at a concentration of 10(-5) M. beta-casomorphin alone did not modify LH basal secretion, but inhibited 25.1 +/- 2.4% (p less than 0.008) LH release enhanced by LHRH. These results indicate that naloxone, an opiate antagonist, markedly increases LH release via a mu-type opioid receptor mechanism at the hypothalamic level only, during short-term exposure.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Melanotropin release inhibiting activity of neuropeptide Y: structure-activity relationships

We have recently shown that the release of alpha-MSH by the intermediate lobe of the frog pituitary is inhibited by neuropeptide Y (NPY). Using the perifusion technique, we have compared in the present study, the alpha-MSH release inhibiting activities of NPY, various NPY short chain analogues and two other members of the pancreatic polypeptide family, peptide YY (PYY) and avian pancreatic polypeptide (APP). The order of biological potency was NPY greater than NPY[2-36] greater than NPY[16-36] greater than NPY[25-36] greater than NPY[1-15]. Among the two pancreatic polypeptides tested, PYY appeared to be almost as potent as NPY while APP was 6 times less active than NPY. Neither NPY[1-15] nor NPY[16-36] could antagonize the inhibitory effect of NPY on alpha-MSH release. The structure-activity relationship study suggests that the bioactive determinant of NPY is located in the C-terminal part of the molecule.     

Effects of VIP, TRH, GABA and dopamine on prolactin release from superfused rat anterior pituitary cells

Effects of VIP, TRH, dopamine and GABA on the secretion of prolactin (PRL) from rat pituitary cells were studied in vitro with a sensitive superfusion method. Dispersed anterior pituitary cells were placed on a Sephadex G-25 column and continuously eluted with KRBG buffer. Infusion of TRH (10(-11) - 10(-8)M) and VIP (10(-9) - 10(-6)M) resulted in a dose-related increase in PRL release. LHRH (10(-8) - 10(-5)M) had no effect on PRL release. On the other hand, infusion of dopamine (10(-9) - 10(-6)M) and GABA (10(-8) - 10(-4)M) suppressed not only the basal PRL release from dispersed pituitary cells but also the PRL response to TRH and VIP. The potency of TRH to stimulate PRL release is greater than that of VIP, and the potency of dopamine to inhibit PRL secretion is stronger than that of GABA on a molar basis. These results indicate that TRH and VIP have a stimulating role whereas dopamine and GABA have an inhibitory role in the regulation of PRL secretion at the pituitary level in the rat.     

Receptor-mediated endocytosis of transferrin in K562 cells

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

Simple method for the detoxification of wastewater ultrafiltration concentrates for rotavirus assay by indirect immunofluorescence.

A simple method for the detoxification of ultrafiltration concentrates of wastewaters for rotavirus assay by the indirect immunofluorescence technique has been developed. Polyacrylamide (Bio-Gel) or dextran (Sephadex G50) beads were mixed with concentrates (0.5 g/10 ml, wt/vol) of wastewaters seeded with simian rotavirus SA11 and allowed to stand for 2 h. The supernatant was decontaminated with antibiotics and then assayed for rotaviruses. Concentrates from raw sewage and treated effluents seeded with SA11 were used to infect MA104 or LLC MK2 cell lines. The concentrates, particularly those from raw sewage and anaerobic waste stabilization ponds, were very toxic to the tissue culture cells. These toxic effects were determined by the detachment and subsequent loss of cells after incubation with concentrates and assay medium for 24 h. They were either completely eliminated or were reduced by greater than 80% after treatment with beads.     

Large-scale purification of plasmid insert DNA sequences using low-percentage agarose exclusion chromatography

Linear double-stranded DNA fragments ranging from 0.14 to 8.4 kbp have been fractionated on low-percentage agarose exclusion gels. Both Ultragel A2 (2% agarose) and Bio-Gel A150m (1% agarose) exclude DNA fragments greater than 900 bp, while the exclusion limit of Bio-Gel A50m (2% agarose) is about 350 bp. All gels result in moderate resolution of DNA fragments smaller than the exclusion limits; we generally observe nearly complete one-step separation of fragments that differ in size by a factor of 2. On the basis of these results, we have used these exclusion gels to routinely purify greater than 0.4 mg of plasmid insert DNA sequences in one step and over 2.5 mg with a single column, demonstrating that these gel matrices can be ideally suited for repeated rapid large-scale purification of plasmid inserts. In addition, this knowledge allows for a more rational design of plasmids in those cases where large-scale use of the insert DNA is required.