Distribution of urocortin 2 in various tissues of the rat.

Urocortin (Ucn) 2 is a new member of the corticotrophin-releasing hormone (CRH) neuropeptide family that is expressed in the central nervous system and peripheral tissues. However, the expression levels of Ucn 2 in various tissues of the rat remains unclear. Thus, the aim of the present study was to characterise the expression of Ucn 2 in the various tissues of the rat. Reverse transcriptase-polymerase chain reaction analysis demonstrated that Ucn 2 mRNA is expressed in the hypothalamus, pituitary, adrenal, stomach, skin, ovary, uterus and skeletal muscle. Histologically, Ucn 2 mRNA and Ucn 2-like immunoreactivity (LI) were demonstrated in both the anterior and intermediate lobes of the pituitary, but not detected in the posterior lobe. Furthermore, all Ucn 2-positive cells in the anterior and intermediate lobes were also positive for beta-endorphin. Ucn 2 mRNA was detected in the adrenal cortex and medulla although Ucn 2-LI was only found in the adrenal medulla. High-performance liquid chromatography analysis of hypothalamic, pituitary, and adrenal extracts showed that the main Ucn 2-LI peak occurred at the same molecular size as that of synthetic Ucn 2. These results suggest that Ucn 2 is synthesised in various tissues, including the anterior and intermediate lobes of the pituitary and the adrenal.

VIP- and PACAP-induced salivary chromogranin A secretion in the isolated perfused submandibular gland of rats.

In the study reported in this paper, sensitive ELISA for rat CgA was developed using synthetic rat CgA(359-389) as antigen, N alpha-biotinylated glycylglycyl rat CgA(359-389), and antirat CgA(359-389) serum for the measurement of CgA-LI in rat saliva. CgA-LI in rat submandibular tissues and saliva was characterized by both immunohistochemical and immunochemical methods. Using isolated perfused rat submandibular gland. VIP at 0.1-1.0 nM in the presence of 0.1 microM ACh was found to cause CgA-LI secretion, whereas neither PACAP-27 nor PACAP-38 showed any effect on CgA secretion.

In-vivo distribution and erythrocyte binding characteristics of cyclosporin in renal transplant patients.

The pharmacokinetic parameters of cyclosporin, a potent immunosuppressive agent, show large intra- and inter-individual variability, possibly because of the different analytical methods used. A recently developed cyclosporin-specific radioimmunoassay has been used to study the in-vivo distribution and binding characteristics of cyclosporin in whole blood, plasma and erythrocytes of fifteen renal transplant patients. The profiles of cyclosporin concentration-time curves after an oral dose of cyclosporin had either one peak (ten patients, group A) or two (five patients, group B). Essentially no difference was observed between the two groups in the relationship between equilibrium cyclosporin concentrations in erythrocyte and plasma as a function of whole-blood concentration. The equilibrium in-vivo cyclosporin concentrations in erythrocyte and plasma were, however, markedly lower than those previously observed under in-vitro conditions. The ratio of cyclosporin concentration in erythrocytes (CE) to that in plasma (CP) changed with time, in inverse proportion to the change in cyclosporin concentration in blood, over the range 0.63-2.80 in individual patients with an average of 1.36 +/- 0.07 (mean +/- s.e.m.) for group A and 1.42 +/- 0.23 for group B. The apparent cyclosporin binding affinity (Kd) to erythrocytes under in-vivo conditions averaged 452.2 +/- 47.6 nM (543.5 +/- 57.2 ng mL-1) for group A and 419.4 +/- 41.2 nM (504.1 +/- 49.5 ng mL-1) for group B, whereas apparent cyclosporin binding capacity (Bmax) of the blood cell averaged 0.83 +/- 0.07 nmol mL-1 for group A and 0.78 +/- 0.07 nmol mL-1 for group B. Significantly reduced average Kd (262.7 +/- 40.2 nM or 315.8 +/- 48.9 ng mL-1, P < 0.01) and Bmax (0.56 +/- 0.08 nmol mL-1, P < 0.05) values were observed during the period after Tmax (4-12 h after the drug ingestion) in group A patients. Apparent Kd and Bmax, determined by a nonlinear regression technique, were 131.6 +/- 29.4 and 1088.0 +/- 114.7 nM (158.2 +/- 35.4 and 1307.8 +/- 137.9 ng mL-1) and 0.178 +/- 0.024 and 0.814 +/- 0.078 nmol mL-1, respectively, during the 4-12 h period in group A patients. These findings reveal distinct differences in in-vivo distribution of cyclosporin and the binding characteristics of the compound to erythrocytes from those previously observed under in-vitro conditions. The significantly lower Kd of cyclosporin binding to erythrocytes during the elimination phase suggests a potential effect of cyclosporin-containing erythrocytes or of cyclosporin contained in erythrocytes during cyclosporin treatment.

Mapping of cyclosporin A binding sites in cyclophilin A by using synthetic peptides.

In order to map cyclosporin A (CsA) binding sites of cyclophilin (CyP), we synthesized the complete set of overlapping 157 octapeptides corresponding to human CyP A using the multi-pin peptide synthesis system. The pin-coupled synthetic octapeptides were examined in terms of binding ability to CsA by a modification of the enzyme-linked immunosorbent assay. Significant binding of CsA was detected with 35 synthetic N alpha-acetylated octapeptides possessing the N-terminal amino acids corresponding to the residues in positions 24-26, 42-44, 69-73, 75, 76, 89-91, 102, 116, 124-131, 144-151 and 152 in human CyP A, respectively. Other eight octapeptides showed moderate CsA binding activity. The distinct binding of octapeptides covering the C-terminal region of the CyP A was particularly significant. These data are to be compared with the information provided by X-ray and NMR studies on the CsA binding sites and furnish thus a test of the reported method. The present study also gave added insight into the CsA interaction sites of CyP.

Gel chromatographic analysis of cyclosporin and its metabolites in human blood compartments.

Gel chromatography combined with specific and non-specific cyclosporin radioimmunoassays was adopted for quantitative analysis of cyclosporin and metabolites in free and protein-bound forms in blood compartments of kidney transplant patients. The analytical method was proved to be useful for the purpose, although plasma protein-bound forms of neither cyclosporin nor metabolites could be quantitated in the system. The present study also provided, by gel chromatographic analysis, additional examples to prove that concentrations of cyclosporin metabolites in blood compartments may not be deduced or inferred simply from those of cyclosporin.

Immunohistochemical localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the adrenal medulla of the rat.

Localization of PACAP in rat adrenal glands was examined by light and electron microscopic immunohistochemistry using a specific antiserum to PACAP 38, R0831. In the light microscopic study, PACAP immunoreactivity was observed in some cell groups in the medulla, but not in the cortex. In comparison with adjacent sections stained with antisera to catecholamine synthesizing enzymes, PACAP-positive cells were immunoreactive to tyrosine hydroxylase and dopamine beta-hydroxylase, but not to phenylethanolamine-N-methyltransferase, suggesting that they were coincident with noradrenaline secreting cells. In the electron microscopic study using the ABC method, DAB reaction products were diffusely distributed in the cytoplasmic matrix of PACAP-positive cells, without intense accumulation on the secretory granules. The splanchnic nerve terminals were PACAP negative. In postembedding immunohistochemistry, gold particles were localized diffusely in the cytoplasma, but not aggregated on the secretory granules. It was suggested that PACAP would localize in the cytoplasmic matrix of noradrenaline cells and stimulate the catecholamine synthesis and release in the adrenal medulla.

Localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats: light and electron microscopic immunocytochemical studies.

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1-38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.

Galanin analogues: agonist and antagonist.

23 galanin-related peptides were synthesized by solid phase technology or conventional solution method. The purity of the products was carefully assessed by routine analytical criteria. Using these synthetic peptides, we have investigated the effects of galanins and structurally modified galanin peptides on glucose-stimulated insulin release using the isolated perfused rat pancreas, gastrin and somatostatin release using the isolated perfused rat stomach, the neurally-evoked muscle contractions in guinea pig ileum and the C-fiber response in the isolated spinal cord of the new born rat. The results suggest that the galanin amino-terminal 1-15 sequence is crucial for its activity in the above four systems. With the goal of developing a specific antagonist of galanin, synthetic galanin (1-15) analogues [D-Thr6,D-Trp8,9]galanin(1-15)ol, and [D-Trp8,9]galanin(1-15)ol were found to be a potent antagonist for inhibitory effect of galanin on glucose-induced insulin release.

[Drug compliance in the elderly].

In order to clarify the characteristics of elderly patients concerning their attitudes toward taking prescribed medicine, self-reported compliance with prescriptions was compared among different age groups. We performed a survey in 626 outpatients and their attending physicians in 4 of our affiliated hospitals, and analyzed self-reported compliance by the patients to the prescription and their answers to questions related to drug-taking along with the diagnoses and prescriptions reported by the physician. The number of prescribed medicine was 2.3 tablets on the average for patients younger than 40, while 5.1 tablets were prescribed for patients over 70. However, self-reported compliance was best in patients over 70 than in other age groups. Compliance was good in 76% of patients who answered that the amount of medicine was appropriate, while good compliance was lower in those who thought the prescription excessive (67%). Likewise, compliance of patients who had concerns with drug side effects or who were not feeling well under medication was lower than that of patients who felt well under medication. Prescriptions for after lunch were most liable to be forgotten than those for other times of the day. Moreover, a high percentage of elderly patients attended more than 2 departments or medical facilities, and one third of those patients did not inform the physician of the fact suggesting that they were at higher risks of overdosing and unexpected drug interaction. Furthermore, the percentage of patients who did not receive explanation from physicians was higher in the elderly thus demonstrating that elderly patients have quite different characteristics in attitude with regard to medicine from younger age group patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin-induced alteration of electrolyte transport in the rat intestine.

Effects of rat and porcine galanin on rat intestinal ion transport were examined in vitro. In the rat distal colon, a sustained increase in short-circuit current (Isc) was produced by the serosal addition of rat galanin at a concentration as low as 10(-9) M, and a maximal increment was observed at 10(-7) M. Porcine galanin was approximately 100 times less potent than rat galanin. In the rat jejunum, rat galanin produced only a slight and transient decrease in basal Isc. The response to rat galanin was not influenced by atropine, hexamethonium, or amiloride, but was virtually abolished by tetrodotoxin or furosemide. Rat galanin did not significantly influence the increase in Isc elicited by electrical field stimulation in the rat colon and jejunum. Transmural unidirectional 22Na and 36Cl fluxes in the rat colonic mucosa were measured under short-circuited conditions, and rat galanin significantly decreased net sodium and net chloride absorption. These findings suggest that galanin acts as a secretory modulator in the rat colon via noncholinergic neural transmission.

[Structure-function studies of galanin].

Galanin is widely distributed in the central and peripheral nervous system and exerts a variety of physiological effects. This review briefly describes the chemical structure, tissue distribution, physiological effects, receptors and structure-function relationships of galanin. It is worth noting that the inhibitory effect of newly synthesized galanin (1-15)-ol on guinea pig ileum contractions was of the same magnitude as that of galanin. This observation gives us an important clue as to the discovery of antagonists of galanin for neural systems.

Gonadotropin-releasing hormone-associated peptide immunoreactivity in bovine colostrum.

Gonadotropin-releasing hormone(GnRH)-associated peptide (GAP) is a 56-amino acid peptide found on the C-terminal of the GnRH (also called luteinizing hormone-releasing hormone) precursor and is assumed to be co-produced with GnRH. The purpose of this report is to demonstrate the presence of GAP immunoreactivity in bovine colostrum. Radioimmunoassay of acidified methanolic extracts demonstrated a concentration of GAP immunoreactivity of approximately 1.5 +/- 0.1 pmol/g dry skim bovine colostrum. Gel filtration (Sephadex G-10) and high-performance liquid chromatography of extracts containing GAP immunoreactivity showed it to be of low molecular weight and a high hydrophobic character. The presence of GAP immunoreactivity in bovine colostrum suggests that the GnRH precursor is synthesized and processed in mammary tissue itself.

Epidermal growth factor and its receptors in human pancreatic carcinoma.

The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells.