Dual effect of verapamil on K-evoked release of endogenous dopamine from arcuate nucleus-median eminence complex

The effect of verapamil, a calcium-entrance blocker, on K⁺-evoked release of endogenous dopamine from tuberoinfundibular neurons incubated in vitro was studied. This compound, added to the incubation medium, at the dose of 10⁻⁶ M, significantly reinforced K⁺-induced dopamine release, whereas, at higher doses (10⁻⁵, 5 × 10⁻⁵ and 10⁻⁴ M), it completely prevented the stimulated dopamine release. The results obtained with the higher doses showed the calcium dependence of K⁺-evoked release of endogenous dopamine from central neurons. The opposite effect, seen with the lower dose of verapamil, could be due to different pharmacological properties of the drug.     

Glutamine-induced membrane currents in cultured chick spinal cord neurons

The effects of -glutamine (GLN) on cultured spinal cord neurons from the chick were studied in the whole cell mode of the patch clamp technique. GLN induced membrane currents rectified at positive membrane potentials (m.p.) and reversed polarity close to zero m.p. The dose-response curve was nearly linear at a semilogarithmic scale for concentrations of 10⁻⁵ M-10⁻² M. Summation of the responses evoked by GLN (10⁻³ M) and glycine (10⁻³ M) was observed when these two amino acids were applied together, while no significant increase of the responses was present when GLN was applied together with -glutamate (10⁻³ M) or kainate (10⁻³ M). It is suggested that GLN binds to the glutamate receptors and activates the same type of ionic channels as glutamate and kainate.     

Lack of effect of opioid compounds on angiotensin II responses of bovine adrenal medullary cells

Angiotensin II (10 nM) increased basal adrenaline and noradrenaline secretion from cultured bovine adrenal chromaffin cells by 2.5- to 3-fold and 4- to 6-fold, respectively, and stimulated basal accumulation of inositol phosphates more than 2-fold. Etorphine and diprenorphine in the range 10⁻⁹ to 10⁻⁵ M had no effect on the catecholamine secretion induced by angiotensin II, and, at 10⁻⁸ and 10⁻⁵ M, had no effect on angiotensin II-induced inositol phosphate accumulation. The functions of adrenal medullary opioid receptors remain to be determined.     

Intracellular Ca antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca²⁺ than in its presence in perfused cat adrenal glands. The intracellular Ca²⁺ antagonist, TMB-8 (10⁻⁴ M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10⁻⁴ M) in the presence of hexamethonium (10⁻³ M) during perfusion with Ca²⁺-free Locke solution containing EGTA (10⁻⁵ M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca²⁺ from an intracellular pool just as caffeine does.     

In vitro effects of adrenomedullin and calcitonin gene related peptide on the release of serotonin and amino acids from rat dorsal spinal cord

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are structurally related, interact with each others receptors and show overlapping biological activities. Immunoreactivity (IR) and mRNAs along with binding sites for both CGRP and adrenomedullin have been shown in the rat spinal cord. CGRP mediates the transmission of nociceptive information at the spinal cord level and both peptides has shown to induces c-fos expression and accumulation of cAMP in spinal cells. In this study, HPLC methods were used to investigate the effects of AM and CGRP on the basal and K⁺-evoked release of serotonin, glutamate (Glu), aspartate (Asp), glycine (Gly) and γ amino butyric acid (GABA) from the slices prepared from the rat spinal cord. Neither CGRP (10⁻⁷ and 10⁻⁶ M) nor AM (10⁻⁷ and 10⁻⁶ M) had significant effects on the basal release of serotonin and the amino acids tested in this study. However, CGRP produced statistically significant increases in the K⁺-evoked release of Asp and Glu, whereas AM failed to do so. Neither AM nor CGRP (10⁻⁷ and 10⁻⁶ M) showed any significant effects on the K⁺-evoked release of serotonin, GABA and Gly. Present data suggest that the stimulatory effects of CGRP on the release of Asp and Glu were exerted by distinct types of CGRP receptors.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

Lack of stimulatory effect of dienogest on the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by endothelial cell as compared with other synthetic progestins

Objectives: Monocyte adhesion to endothelial cells is an important initial event at the onset of atherosclerosis. It is partially mediated by the expression of adhesion molecules on the endothelial cell surface. While estrogens inhibit the development of atherosclerosis, the effect of co-administered progestin remains controversial. We examined the effect of progestins on cytokine-stimulated human umbilical venous endothelial cell (HUVEC) expression of adhesion molecules. Methods: In HUVECs, mRNA expression of progesterone receptors (PRs) and androgen receptors (AR) was determined by RT-PCR. HUVECs were stimulated by interleukin-1β (IL-1β) for 24 h with or without various steroids, and then the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was semiquantified by ELISA. Results: In all preparations of HUVECs used in this study, RT-PCR confirmed mRNA expression of both isoforms of PR, PR-A and PR-B, as well as AR. Addition of progesterone (10⁻¹⁰–10⁻⁷ M) or dienogest (DNG) (10⁻¹⁰–10⁻⁸ M) did not affect IL-1β-stimulated ICAM-1 or VCAM-1 expression. In contrast, medroxyprogesterone acetate, norethindrone acetate and levonorgestrel (10⁻¹⁰–10⁻⁸ M) dose-dependently increased cell adhesion molecules. The progestin-induced increase was blocked by the concomitant addition of mifepristone, a PR antagonist, but not by hydroxyflutamide, an AR antagonist, indicating that the progestin stimulation was mediated predominantly via PR. Conclusions: These results suggest that DNG, unlike other synthetic progestins, lacks stimulation of cell adhesion molecules. For the prevention of atherosclerosis, estrogen in combination with DNG may be a suitable regimen in hormone replacement therapy in postmenopausal women.     

Characterization of CD28CD4 T cells in living kidney transplant patients with long-term allograft acceptance

CD28⁻CD4⁺ T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28⁻CD4⁺ T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V_β repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28⁻CD4⁺ T cells increased correlatively with time after transplantation in this group of patients. The CD28⁻CD4⁺ T cells did not express detectable levels of CD25, CD69, V24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28⁻CD4⁺ T cells revealed a restricted V_β repertoire, whereas the V_β usage of CD28⁺CD4⁺ T cells from the same patients was much diversified. Expression levels of TGF-β and IFNγ gene were significantly higher in the CD28⁻ CD4⁺ T cells than in the CD28⁺CD4⁺ T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28⁻ CD4⁺ T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.     

Acetylcholine receptors in dissociated nucleus basalis of Meynert neurons of the rat

Electrical and pharmacological properties of acetylcholine (ACh)-induced currents in neurons dissociated from the nucleus basalis of Meynert (nBM) of immature (2-week-old) rats were investigated with the whole-cell mode of the patch-clamp technique. At a holding potential (V_H) of −50 mV, ACh (10⁻⁴M) evoked a transient inward current mimicked by nicotine (I_nACh), followed by a sustained outward current mimicked by carbamylcholine (I_mACh). The K_D values were 1.2 × 10⁻⁴ M for I_nACh) and 8.7 × 10⁻⁷ M for I_mACh. The reversal potenial of I_mACh was close to E_K. The I_mACh was determined to be elicited via the M₂ muscarinic receptor, based on the differences in sensitivity to muscarinic antagonists such as pirenzepine and AF-DX-116.     

Human suppressor T cells: Induction, differentiation, and regulatory functions

T-cell-mediated suppression of human immune responses involves a complex interaction between distinct lymphocyte subsets with suppressor-inducer and suppressor-effector functions. Recent studies with subset-specific monoclonal antibodies have defined a characteristic phenotype of suppressor-inducer cells (CD4⁺ Leu8⁺ 2H4⁺ 4B4⁻) that can be distinguished from that of helper cells for antibody synthesis (CD4⁺ Leu8⁻ 2H4⁻ 4B4⁺). Similarly, suppressor-effector cells (CD8⁺ CD11⁺ Tp44⁻ can typically be defined as a subset separable from cytotoxic T cells (CD8⁺ CD11⁻ Tp44⁺). Both antigen-specific and nonspecific interactions are important in suppressor T-cell activation and function. Soluble signals required for differentiation of CD8⁺ suppressor cells include an indomethacin-sensitive monocyte product and interferon gamma. In contrast, proliferation of the CD8⁺ suppressor cell subset depends on stimulations first by a product of CD4⁺ Leu8⁺ cells, T suppressor cell growth factor, and second by interleukin 2. Although the molecular basis of antigen-specific interactions between CD4⁺ and CD8⁺ cells in suppressor cell generation has not been defined, it may involve both conventional, presumably MHC-restricted, interactions between antigen and antigen receptors, as well as anti-idiotypic interactions of suppressor-effectors with determinants on suppressor-inducer receptors. Progress in elucidating requirements for activation, growth, and differentiation of suppressor cells should facilitate long-term culture of such cells and lead to clearer understanding of mechanism of suppressor-cell mediated immunoregulation.     

Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase

Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37°C, 10⁻⁵ M or 10⁻⁶ M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10⁻⁵ M also significantly decreased the rate of ageing in 150 mM NaCl. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.     

Cell mediated immunity changes in ageing, relative importance of cell subpopulation switches and of nutritional factors

Decreased T-cell functions with ageing have been extensively described. This review focuses on recent data on changes in T-cell subpopulations related to ageing and their consequences on T-cell proliferation. Increase of immature T cells CD2⁺ CD3⁻ is an ageing phenomenon related to T-cell declining proliferation. Recently it was shown that increase of immature T cells was due to an increase in different subtypes of the CD2⁺ CD3⁻ population, double-negative CD2⁺ CD4⁻ CD8⁻ and double-positive CD2⁺ CD4⁺ CD8⁺ subpopulations, the former being associated with nutritional deficit, the latter with associated diseases. Other authors have focused on decreases of naive T cells with parallel increase of memory T cells; such a switch is also relevant to declining T-cell proliferation. This review focuses on two major factors which influence immune ageing: nutritional parameters and antigen exposure.     

A quantitative analysis of rat adrenal chromaffin tissue: Morphometric analysis at tissue and cellular level correlated with catecholamine content

A morphometric analysis of normal Wistar rat adrenal medulla following perfusion fixation and Araldite embedding, was correlated with catecholamine levels on fresh tissue, measured by high-performance liquid chromatography. The mean volume of whole adrenal is 13.2 mm³ and the mean medullary volume 1.3mm³. Volume density estimates showed that the medulla is composed of 63% chromaffin tissue with an adrenaline to noradrenaline storing cell ratio of 4.4:1. The vasculature occupies 20%, neuronal tissue 5% and interstitial tissues 12% of the medulla. A comparison was made of cell volumes, cell numbers and volume and surface density estimates of cytoplasmic organdies in adrenaline and noradrenaline storing cells. The mean cell volume of adrenaline storing cells at 1300 μm³ is larger than that of noradrenaline storing cells at 980 μm³. A single adrenal medulla contains4.4−5.7 × 10⁵ adrenaline cells and1.5−1.9 × 10⁵ noradrenaline cells. Chromaffin granules account for approximately 30% of the volume of the cytoplasm; the numerical density of granules at different sites in the cell was calculated for adrenaline cells. The volume density of mitochondria (4%) and the surface density of mitochondrial membranes (the ratio of outer to inner membrane being approximately 1:2.3) were similar in both cell types. Rough endoplasmic reticulum was the only organelle to show a significant difference in volume and surface density between the two cell types. Adrenaline storing cells have stacks of rough endoplasmic reticulum which have two to three times the surface and volume densities of that found diffusely scattered throughout noradrenaline cells. The adrenaline content of an adrenaline storing cell is0.14 × 10⁻⁶ μM and that of a granule 3.0 × 10⁻¹² or3.8 × 10⁻¹² μ moles depending on the method of calculation. The noradrenaline content of noradrenaline storing cells can only be calculated on the assumption that all noradrenaline is stored in this cell type though it is likely that some is contained within adrenaline cells. Based on this assumption the noradrenaline content is0.17 × 10⁻⁶μ moles per cell and5 × 10⁻¹² μ moles per granule. The present study provides baseline morphometric data on the rat adrenal medulla at tissue and cellular level correlated with amine levels in adrenaline and noradrenaline storing cells and granules.     

Hydrocortisone stimulation of DNA synthesis in bromodeoxyuridine-selected non-dividing WI-38 cells

Ostensibly noncycling WI-38 cells were selected by incubating growing cultures for 7 days with 10⁻⁵ M bromodeoxyuridine. These cells were then exposed to Hoechst dye 33258, and then visible light which kills cells that incorporated bromodeoxyuridine. Following selection, cultures were refed with medium containing 10% fetal bovine serum. By autoradiography, we determined that less than 10% of these cells could incorporate [³H] thymidine during the next 7 days. This value was increased to 25% or more by adding hydrocortisone to the medium. The proliferative response was also increased by refeeding cultures with hydrocortisone-containing medium conditioned 24 h by freshly seeded mid-, but not late, population doubling level (PDL) cultures. Medium conditioned without hydrocortisone did not stimulate incorporation of [³H] thymidine beyond the control value. These results show that: (1) cells that might otherwise not initiate DNA synthesis are stimulated to do so by hydrocortisone; (2)hydrocortisone-conditioned medium from mid-PDL culture increases this stimulation; and (3) hydrocortisone-conditioned medium from late PDL cultures is not stimulatory.     

Cell generation within human thymic subsets defined by selective expression of CD45 (T200) isoforms

Although the thymus is the source of all mature peripheral T lymphocytes, the majority of thymocytes die intrathymically. Until recently, there has been no phenotypic marker to allow definition of the generative thymocyte lineage, thereby distinguishing those thymocytes committed to death from those which will evenually give rise to thymic emigrants. We believe that expression of the high-molecular-mass isoforms (p190, p205, and/or p220) of the leukocyte common antigen (CD45) distinguishes the thymic generative lineage from the vast majority of thymocytes expressing the low-molecular-mass isoform (p180) of CD45 and committed to die within the thymus. The thymocytes defined by their lack of CD45 p180, the low-molecular-mass isoform, comprise all thymocytes with clonogenic potential and include all major subsets defined by CD4 and CD8. We have proposed that a CD45 p180⁻ lineage exists in the human thymus and that this lineage results in the production of mature thymocytes and thymic emigrants. The objective of the present study was to determine by DNA analysis whether the degree of cell cycling in subsets of human thymus, defined by selective expression of high-molecular-mass isoforms of CD45, was sufficient to account for the generation of thymic emigrants. Multicolor immunofluorescence analysis of surface markers and 7-amino actinomycin D as well as propidium iodide staing was used to measure the DNA content of thymic subsets. Negative depletion methods were used to isolate and characterize human thymocyte subsets defined by CD45 isoform, CD3, CD4, and CD8, and subsequently to determine the cell cycle status of the isolated subsets by flow-cytometric analysis of cellular DNA content. CD3^−/lo thymocytes had a high number and CD1^−/lo thymocytes a low number of cycling cells, consistent with murine data. CD45 p 180⁻ cells, as well as the CD4⁻8⁻ and CD3⁻4⁻8⁻ subsets which express high molecular-weight CD45 isoforms, exhibited a significant number of cycling cells. Since CD45 p180- thymocytes exhibited a significant number of cycling cells, based on numerical arguments we conclude that this cycling thymocyte fraction is capable of generating the daily requirements of mature thymocytes and thymic emigrants.     

Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas

In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca²⁺ and Mg²⁺ in fusion medium and pretreatment of mixed cells with proteases improved hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 × 10⁻⁴ and 1 ×10⁻⁴, respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%–50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.     

Mouse antibody production test: can we do without it?

Introduction of microbiologically contaminated materials into mice can cause infections of the recipients and jeopardize experimental protocols. As such, the methods used to screen biological materials should be sensitive, reliable and suitable for routine diagnostic work. In this report, the sensitivity of the viral plaque assay, mouse antibody production test and polymerase chain reaction (PCR) for detection of MHV-A59 and MMVp, two of the most prevalent pathogenic viruses in experimental mouse facilities, was compared. Analysis of serial tenfold dilutions of virus stocks revealed that the sensitivity of the mouse antibody production test on day 28 (10⁻¹⁰ dilution) was at least 10 times higher than that of the viral plaque assay (10⁻⁹ dilution) and 10⁴ times more than that of the RT-PCR (10⁻⁶ dilution) for detection of MHV-A59. For detection of MMVp, the PCR (10⁻¹⁰ dilution) proved to be 10⁶ times more sensitive than the viral plaque assay (10⁻⁴ dilution) and the mouse antibody production test on day 28 (10⁻⁴ dilution) which were equally sensitive. Based on the present study, it was shown that the method for diagnosis of viruses in biological materials should be employed only after the sensitivity has been determined for the viruses of interest implying that the most sensitive method needs to be determined independently for each virus.     

UV-B and lipid hydroperoxides promote conjunctival epithelial cell migration

The idea that sunlight itself may be a major risk factor in certain eye diseases has been widely accepted. Peroxides or radicals arising from UV irradiation may induce factors leading to cell migration. To clarify this relationship further, we have examined the effects of UV radiation and lipid hydroperoxides on cell migration of conjunctival epithelial cells in culture from palpebral, fornical and bulbar regions. The cell migration from each region was enhanced by UV-B irradiation at 50 mJ/cm² and linoleic acid hydroperoxide (LHP) at 10⁻⁷ M concentration. In contrast, cell migration was suppressed by UV-B irradiation at 100 mJ/cm² and LHP at 10⁻⁵ M. Both UV-B and LHP induced cell migration was also suppressed by a vitamin E and C conjugated antioxidant (EPC-K1) which has radical scavenging activity. These data suggest that reactive oxygen intermediates play a prominent role in cell migration.     

Phenotypic development of neonatal rat chromaffin cells in response to adrenal growth factors and glucocorticoids: focus on pituitary adenylate cyclase activating polypeptide

We have investigated the regulation of the morphological phenotype of chromaffin cells cultured from 6-day-old rat adrenal glands. We show that pituitary adenylate cyclase activating polypeptide (PACAP), which is present in and released from nerves innervating chromaffin cells, rapidly induces neuritic growth, affecting 25% of tyrosine hydroxylase-positive chromaffin cells after 3 days at an optimal concentration of about 20 nM. PACAP does not synergistically act with other factors known to promote neurite growth, including nerve growth factor (NGF), basic fibroblast growth factor (bFGF, FGF-2), and ciliary neurotrophic factor (CNTF). The neurite promoting effect of PACAP and FGF-2 is entirely overridden by dexamethasone (2 × 10⁻⁸ M) suggesting that, despite the presence of these promoting factors in the adrenal medulla, glucocorticoids from the adrenal cortex are probably sufficient to prevent the development of neuronal traits in adrenal chromaffin cells.