Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography

A sensitive and selective method for the determination of four threonine (Thr) isomers (L-Thr, D-Thr, L-allo-Thr and D-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of D-Thr, D-allo-Thr and L-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol-5 pmol per injection, and that of L-Thr is 50 fmol-50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of D-Thr, D-allo-Thr and L-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of D-Thr and D-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of D-Thr (0.85 +/- 0.05 nmol/g wet tissue) and D-allo-Thr (5.01 +/- 0.32 nmol/g wet tissue) were found in the corpus striatum. L-allo-Thr was not present in any of the tested tissues and physiological fluids.     

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.     

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

Circadian changes of D-alanine and related compounds in rats and the effect of restricted feeding on their amounts

The circadian changes of D-alanine (D-Ala), an intrinsic D-amino acid found in mammals, were investigated in rats with diurnal and nocturnal habits, and the profiles were compared to those of L-Ala, other D-amino acids and several hormones. Determination of D-Ala in the rat plasma, pancreas and anterior pituitary gland was carried out using a sensitive and selective two-dimensional HPLC system combining a micro-ODS column and an enantioselective column after fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The amount of D-Ala was high during the sleeping period and low during the active period in rats with both diurnal and nocturnal habits, indicating for the first time that the D-Ala is closely related to the activity rhythm of animals. In contrast, L-Ala and other D-amino acids did not show any clear circadian changes. The circadian change of D-Ala inversely correlated with that of the plasma insulin level in rats with both diurnal and nocturnal habits. Considered together with our previous findings that D-Ala is localized in the insulin secreting beta-cells in the rat pancreas, it is strongly suggested that D-Ala has some functional relationships to insulin in mammals.     

D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea

Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.     

d-Alanine in the islets of Langerhans of rat pancreas

Relatively high levels of d-alanine (d-Ala), an endogenous d-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed d-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180 ± 60 fmol d-Ala per islet (300 ± 100 nmol/g islet), and 10 ± 2.5 nmol/g of wet tissue in pituitary. Additionally, 12 ± 5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of d-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in d-Ala release of 0.6 ± 0.5 fmol per islet. As d-Ala is stored in islets and released in response to changes in extracellular glucose, d-Ala may have a hormonal role.     

Specific binding of 125I-LHRH agonist to hippocampal membranes: fluctuations during the estrous cycle.

Specific binding of 125I-[D-Ala6-CH3-Leu7-Pro9,NHET]LHRH, a LHRH agonist, to hippocampal membranes prepared from ovariectomized female rates was examined. One high affinity binding site was observed with a Kd of 0.12 +/- 0.01 nM and an apparent Bmax of 13.0 +/- 3.8 fmol/mg. Luteinizing hormone-releasing hormone and the behaviorally active Ac-LHRH(5-10) were able to compete for the agonist binding site. Native LHRH had an apparent Ki of 1.73 nM, while AC-LHRH(5-10) was 30 times less potent. Competition studies examined over the rat estrous cycle revealed an eighteenfold decrease in apparent affinity during diestrus I and estrus compared with ovariectomized animals. Tissue from animals in proestrus had a Ki of 5.0 nM. Specific binding studies indicate that receptor concentration is highest in proestrus (6.11 +/- 0.90 fmol/mg) and significantly lower during estrus (2.4 +/- 0.29 fmol/mg). These data suggest that at least one fragment of native LHRH can interact with neuronal LHRH receptors and that these receptors, like those in the pituitary, can be modulated by circulating steroid hormones.     

Regional Distribution and Characterization of Kinin in the CNS of the Rat

The distribution of kinin in the CNS of the rat, which was extracted with n-butanol from an acidified homogenate, was determined using a bradykinin (BK) radioimmunoassay system. The immunoreactive kinin was widely distributed throughout the brain. The highest content was found in the pituitary gland (4,135 fmol BK Eq/g), followed by the medulla oblongata (912 fmol/g), cerebellum (549 fmol/g), and cortex (512 fmol/g). The kinin in the posterior pituitary was concentrated 4.5 times as much as in the anterior lobe. Serial dilution of brain extracts produced binding curves parallel to the standard radioimmunoassay curve. The purified brain kinin comigrated with authentic BK during CM-cellulose chromatography and Sephadex LH-20 gel chromatography. Its molecular weight was estimated to be 1,127 +/- 45 by gel filtration, which coincides well with that of BK. Chymotrypsin degraded the extracted kinin and authentic BK, but trypsin did not. These data demonstrate that a peptide indistinguishable from BK exists in the rat brain. Furthermore, pituitary kinin was separated into BK (87%), Lys-BK (10%), and Met-Lys-BK (3%), using reverse phase HPLC.     

[3H]Nitrendipine Binding to Calcium Channels in Bovine and Rat Pituitary

[3H]Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 +/- 0.30 pmol/g wet weight of tissue and the KD was 1.44 +/- 0.02 X 10(-10) M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage-sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 X 10(-10) M, respectively. Verapamil and the verapamil-like calcium channel blockers D-600 and tiapamil competed in a complex manner for the [3H]nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific [3H]nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of [3H]nitrendipine binding in the pituitary tissue resemble strongly the properties of [3H]nitrendipine binding in the brain which is believed to be to voltage-sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.     

Quantitative autoradiographic determination of angiotensin-converting enzyme binding in rat pituitary and adrenal glands with 125I-351A, a specific inhibitor

We describe a quantitative autoradiographic technique which allows measurement of angiotensin-I-converting enzyme [ACE] (kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) levels in discrete areas of pituitary and adrenal glands in individual animals. Tissue sections were incubated with 125I-351A, a specific ACE inhibitor, and results were obtained with computerized densitometry and comparison to 125I standards. There were high levels of ACE in both the anterior and posterior lobes of the pituitary, with no detectable binding in the intermediate lobe. The maximum binding capacity (Bmax) was 920 +/- 62 fmol/mg protein for the anterior pituitary and 1162 +/- 67 fmol/mg protein for posterior pituitary. The binding affinity constant (Ka) was 0.95 +/- 0.11 X 10(9) M-1 and 1.20 +/- 0.19 X 10(9) M-1 for the anterior and posterior lobes, respectively. In the adrenal gland, there were two distinct areas of specific binding, the adrenal medulla and the adrenal capsule-zona glomerulosa area. The Bmax for the adrenal medulla was 652 +/- 80 fmol/mg protein and 294 +/- 53 fmol/mg protein for the adrenal capsule-zona glomerulosa. The Ka for 351A was 1.04 +/- 0.19 X 10(9) M-1 and 1.74 +/- 0.40 X 10(9) M-1 for medulla and adrenal capsule-zona glomerulosa respectively. The results support the existence of local ANG systems active in both the pituitary and adrenal glands.     

Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland

The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [125I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [125I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [125I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland.     

Effects of clomiphene citrate on the pituitary gland in chronically estrogenized rat

Effects of clomiphene citrate (clomiphene) on the pituitary gland of chronically estrogenized ovariectomized rats were investigated. Estradiol-17 beta (E2) pellet implanted subcutaneously in castrated rats for 7 days caused significant increases in pituitary weight and serum prolactin (PRL) level but suppressed serum luteinizing hormone (LH) level. In the estrogenized rats about 40% of estrogen receptor (ER) found in whole pituitary cells (65 +/- 7 fmol/10 mg tissue) was observed in the nucleus, while 60% of ER was present in the cytosol fraction. A single injection of 5 micrograms E2 translocated cytosol ER immediately to nuclear compartment; amounts of ER found in cytosol and nuclear fractions were 16 +/- 1 and 37 +/- 4 fmol/10 mg tissue, respectively, at 1 h. However, the distribution of ER returned to the pre-injection level within 4 h. In the non-estrogenized castrated rats, the nuclear retention of ER was significantly longer than that in the estrogenized rats. A single administration of 200 micrograms clomiphene in the estrogenized rats, on the other hand, increased nuclear ER gradually. Nuclear ER reached the peak level at 4 h (62 +/- 5 fmol/10 mg tissue) and the level remained almost unchanged for 24 h. Cytosol ER decreased and reached a nadir at 4 h (4.3 +/- 0.3 fmol), and the replenishment of cytosol ER could not be detected for 24 h. Similar patterns of cytosol and nuclear ER following the clomiphene injection were also found in the castrated rats. The clomiphene administration in the estrogenized rats resulted in a significant reduction of the pituitary weight 48 h after the administration. The present results seem to show the antiestrogenic action of clomiphene in the pituitary gland.     

Go, a GTP-Binding Protein: Immunochemical and Immunohistochemical Localization in the Rat

The tissue and cellular distribution of a GTP-binding protein, Go, was investigated in the rat by immunochemical and immunohistochemical methods. Because the specific antibody for the alpha subunit of bovine Go (Go alpha) cross-reacted with rat Go alpha, an enzyme immunoassay method developed for bovine Go alpha was applied for measuring the tissue concentration of Go alpha in the rat. Go alpha was detected in all tissues examined except blood cells. The concentration of Go alpha was highest in the CNS (approximately 7.7 and 4.4 nmol/g in the cerebrum and cerebellum, respectively), followed by the pituitary gland and sciatic nerve. Among the other peripheral tissues, relatively high concentrations of Go alpha were observed in the urinary bladder, stomach, and intestines; however, these values were less than 2% of the concentration in the cerebrum. Go alpha in the intestine was located mostly in the muscle layer. Immunohistochemical study showed that Go alpha was associated mostly with the neural elements but not with cells particular to each peripheral organ. Go alpha was also present in the membranes of neuroendocrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, islets cells in the pancreas, and parafollicular cells in the thyroid. These results indicate that Go is localized exclusively in the nervous tissues and neuroendocrine cells.     

Distribution and characterization of immunoreactive prolactin-releasing peptide (PrRP) in rat tissue and plasma

We established a sensitive and specific two-site enzyme immunoassay (EIA) for prolactin-releasing peptide (PrRP) using two region-specific monoclonal antibodies. We investigated the tissue distribution and the plasma concentration of immunoreactive (ir-) PrRP in rats using this assay. Ir-PrRP was widely distributed in the central nervous system and pituitary gland. The highest concentration of ir-PrRP was found in the hypothalamus. In peripheral tissues, appreciable levels of ir-PrRP were found only in the adrenal gland. The mean plasma concentration of ir-PrRP was 0.13 +/- 0.01 fmol/ml (mean +/- SEM). In reverse-phase and gel-filtration high performance liquid chromatography, hypothalamic ir-PrRP eluted at a position identical to that of PrRP31 and PrRP20. On the other hand, ir-PrRP from the adrenal gland and plasma eluted only at the position of synthetic PrRP31, indicating that molecular forms of ir-PrRP in vivo differed among tissues.     

Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone

The relationship between number of receptors for gonadotropin-releasing hormone (GnRH) and the ability of the anterior pituitary gland to release luteinizing hormone (LH) was examined in ovariectomized ewes. A GnRH antagonist was used to regulate the number of available receptors. The dose of GnRH antagonist required to saturate approximately 50 and 90% of GnRH receptors in ovariectomized ewes was determined. Thirty min after intracarotid infusion of GnRH antagonist, ewes were killed and the number of unsaturated (i.e., those available for binding) pituitary GnRH receptors was quantified. Infusion of 10 and 150 micrograms GnRH antagonist over a 5-min period reduced binding of the labeled ligand to approximately 50 and 12% of controls, respectively. The effect of reducing the number of GnRH receptors on release of LH after varying doses of the GnRH agonist, D-Ala6-GnRH-Pro9-ethylamide (D-Ala6-GnRH) was then evaluated. One of four doses of D-Ala6-GnRH (0.125, 2.5, 50 and 400 micrograms) was given i.v. to 48 ovariectomized ewes whose GnRH receptors had not been changed or were reduced to approximately 50 or 12% of control ewes. In ewes with a 50% reduction in GnRH receptors, total release of LH (area under response curve) was lower than that obtained for controls (P less than 0.01) at the 0.125-micrograms dose of D-Ala (6.1 +/- 0.7 cm2 vs. 13.5 +/- 0.7 cm2) but was not different at the 2.5-, 50- or 400-micrograms doses of D-Ala6-GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.