Analysis of the responses of frog motoneurons to epinephrine and norepinephrine

Epinephrine and norepinephrine were applied to the isolated superfused frog spinal cord hyperpolarized motoneurons. The hyperpolarization was related to both direct and indirect actions and the indirect effects were produced by activation of alpha 2-adrenoceptors. In about half of the spinal cords a slow depolarization caused by activation of beta-receptors was seen and was largely attributable to direct actions of the catecholamines on motoneuron membranes. In a small number of preparations an early alpha 1-mediated depolarization was noted. The results suggest that catecholamines released from terminals in the frog ventral horn could exert a modulatory action on the motoneuron output from the spinal cord.     

Receptor desensitization and the transient nature of the secretory response of adrenal chromaffin cells

Transition of the cellular responsiveness to a cholinergic stimulation during a prolonged stimulation with acetylcholine (ACh) of adrenal chromaffin cells was studied by directly monitoring the secretory process in cell bed perfusion experiments. A pulsatile injection of hexamethonium during the prolonged stimulation caused an interruption of the response. Step-up of the ACh concentration in the declining phase of the primary response evoked an additional response. The magnitude of the response to the step-up was reduced by extracellular Ca2+ in a dose-dependent manner. However, neither the peak amplitude nor the declining rate of the primary response was affected by Ca2+ in such a direction. These results suggest that the transient nature of the ACh-evoked response is mainly attributable to the inactivation of transduction mechanisms between receptor stimulation and channel activation. Although Ca2+-facilitated desensitization of nicotinic receptors is in process during the stimulation, such a desensitization may account only insignificantly for the transient nature.     

Opossum adrenal medulla: II. Differentiation of the chromaffin cell

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochro-moblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.     

Acute modulation of adrenal chromaffin cell BK channel gating and cell excitability by glucocorticoids

Although adrenal glucocorticoids cortisol and corticosterone (CORT) have numerous "genomic" effects on adrenomedullary chromaffin cells, acute modulatory actions remain largely unknown, despite rapid stress-related changes in secretion. We report that 1 microM glucocorticoids rapidly modulate gating of chromaffin cell BK channels and action potential firing. In general, CORT, or the analog dexamethasone (DEX), increased channel activity in inside-out bovine patches, an effect not blocked by the glucocorticoid receptor (GR) antagonist RU38486. By contrast, these steroids could profoundly inhibit BK activation in many rat patches, while facilitating activation in others. We show that BK inhibition arises from a negative shift in the voltage dependence of BK inactivation paralleling that for activation. We report that rat cells characteristically exhibit greater repetitive firing ability than bovine cells in the absence of glucocorticoids. In both species, steroid application typically increased firing responses to smaller current injections, attributable to BK-enhanced repolarization and Na+ channel deinactivation. However, in rat cells, where BK inactivation is generally faster and more complete, glucocorticoids tended to dampen responses to stronger stimuli. Thus, in the context of natural variation in BK gating, glucocorticoids can either promote or limit firing responses. We suggest that steroids exploit BK gating variety to tailor catecholamine output in a species- and context-specific fashion.     

The secretory mechanism in adrenal chromaffin cells by nitrophenol compounds: possible involvement of the change in the surface potentials

The rabbit adrenal gland was perfused with a modified Locke's medium and the mechanism of adrenaline secretion induced by nitrophenol compound was investigated. The secretory response to 2,4-dinitrophenol (DNP) or trinitrophenol (TNP) showed the following peculiar features. (1) Prolonged exposure to DNP or TNP caused an immediate, and long-lasting increase of secretion only in the presence of Ca. (2) The response to DNP depended on the concentration of Ca, but that to TNP was largest in the presence of 0.5 mM Ca. (3) Re-addition of 2 mM Ca during prolonged exposure to DNP or TNP produced a larger response than did the simultaneous addition of Ca and either DNP or TNP. (4) The response to DNP in the presence of 2 mM Ca was markedly reduced by removal of external Na, but the Na dependency became less marked in the presence of 0.1 mM Ca. The response to TNP in the presence of 0.5 mM Ca was slightly reduced in the absence of Na. (5) DNP or TNP caused a much larger response during depolarization with a high K, Rb, or Cs medium than that with a 5 mM KCl medium. (6) The response to DNP was largely potentiated in the absence of most ions in the presence of only 0.1 mM Ca and that to TNP was potentiated in the presence of only 0.5 mM Ca under the same condition. (7) Re-addition of Ca 5 min after the removal DNP or TNP still caused substantial secretion. These results suggest that nitrophenol compounds stimulate secretion by two independent mechanisms: one is related to its effect on the surface potentials of the plasma membrane and the other unknown except for the possible dependence of Na and Ca.     

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1–4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca²⁺ influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca²⁺ influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr⁵¹⁴ and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca²⁺ influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.     

Regulation of skeletal muscle development by the central nervous system in the fetal pig

The effect of upper motor neuron regulation on skeletal muscle development was studied in the fetal pig. A region of the spinal cord at the level of the upper cervical vertebrae was destroyed by cauterization at 45 days of gestation in four pig fetuses. Five fetuses with intact spinal cords served as controls. Innervation and enzyme activities in the longissimus muscle, the ultrastructure and quantitation of satellite cells in the sartorius muscle, and plasma composition were evaluated at 110 days of gestation. The terminal innervation ratios were similar (P greater than 0.05) for muscles from control and cauterized fetuses. Endplate morphology was also similar. Therefore, innervation of newly formed primary fibers is not controlled by upper motor neurons after 45 days of gestation. Mean values for body weight, percentage of muscle dry weight, percentage of myofibers with myonuclei and plasma levels of protein, glucose, triglycerides, lactate, and creatine phosphokinase activity were similar (P greater than 0.05) between the two groups of fetuses. All but one muscle fiber examined was of the secondary fiber type. These observations suggest that the physiological maturity of the muscle was not appreciably altered even though glucose-6-phosphate dehydrogenase activity was higher (P greater than 0.05) and total phosphorylase activity was lower (P greater than 0.05) in the spinal cauterized fetuses than in the control group. The percentage of satellite cells was lower when based on the number of myofibers observed (P greater than 0.005) or on the number of nuclei contained within the basal lamina (P greater than 0.001) in the muscle of the spinal cauterized fetuses than in the control fetuses. The cytoplasm of satellite cells from the muscles of control fetuses was rich in organelles indicative of metabolic and mitotic activity whereas a paucity of such organelles was observed in the satellite cells of cauterized fetuses. Since the percentage of myofibers that had myonuclei was similar (P greater than 0.05) for the control and cauterized fetuses, it appeared that the myonuclear population was maintained by direct incorporation of the parent satellite cell.     

Immune cell traffic control and the central nervous system

That lymphocytes traffic to the central nervous system (CNS) is a relatively new concept. From studies on lymphocyte trafficking to lymphoid tissue and systemic organs, it is known that small lymphocytes recirculate continually from blood to lymphoid tissue and back again to blood, and that lymphocyte activation leads to changes in trafficking patterns with rerouting to areas of antigen deposition. Control of lymphocyte trafficking involves the induction and expression of a series of cell-surface molecules on circulating immune cells and target tissue vasculature. Lymphocyte trafficking into the CNS preferentially involves activated lymphocytes. This may be secondary to elaboration by activated immune cells of cytokines which are capable of stimulating CNS endothelium and may also relate to their production of enzymes which degrade cytoskeletal elements, aiding transmigration. Based upon observations of systemic homing receptors specific for certain organs, the existence of CNS-specific homing receptors with corresponding ligands on CNS vasculature is postulated.     

Development of central control of norepinephrine turnover and release in the rat heart: responses to tyramine, 2-deoxyglucose and hydralazine

Cardiac responses to centrally-mediated stimulation of the efferent sympathetic nervous system appear postnatally in the rat. To identify the sequence of maturational events in this pathway, the abilities of selected drugs to elicit cardiac norepinephrine release were evaluated. Tyramine, which displaces norepinephrine from nerve terminals, produced release at the earliest age tested. However, 2-deoxyglucose, which causes centrally-evoked stimulation of the sympathetic nervous system, did not elicit a response until about one week postnatally. Hydralazine lowers blood pressure and thus requires function of both baroreceptor afferent and sympathetic efferent pathways in order to produce stimulation; hydralazine was first able to cause loss of norepinephrine from cardiac sympathetic terminals at 14 days of age.These results suggest that although the noradrenergic nerve terminals in the neonatal myocardium are immature, they do contain some releasable norepinephrine. The failure of 2-deoxyglucose and hydralazine to elicit release indicates that the onset of overall function of this pathway is attendant upon maturation of synapses central to those in the myocardium. Additionally, the spontaneous turnover rate of norepinephrine, measured after alpha-methyl-p-tyrosine administration, was low at birth, showed a peak at two weeks of age, and declined thereafter toward the adult value; the peak corresponded to a transient increase in sympathetic activity inferred previously from indirect measurements, and correlated temporally with onset of baroreceptor control of sympathetic tone.     

Hormonal influence on the secretory immune system of the eye: endocrine interactions in the control of IgA and secretory component levels in tears of rats.

Previous research from our laboratory has demonstrated that androgens regulate the ocular secretory immune system of the rat. The purpose of the present study was to determine whether other hormones might influence this androgen effect. Experiments involved the daily administration of saline or hormones to adult orchiectomized rats, the collection of tears 24 hr after the fourth hormone injection, and the measurement of free secretory component (SC), IgA and total protein levels in tears. Our first aim was to evaluate whether female sex steroids might antagonize androgen action on tear IgA and SC: orchiectomized rats were treated with combinations of saline, testosterone, oestradiol or progesterone. Testosterone induced a significant increase in the tear SC and IgA concentrations, as compared to those of saline-injected controls. This androgen effect was not inhibited by co-treatment with oestradiol or progesterone, nor duplicated by the administration of these hormones alone. Our second aim was to assess whether the absence of certain hormones might alter tear SC and IgA levels, or influence the ocular response to androgen exposure: rats underwent orchiectomies and specific endocrine organ ablations or appropriate sham-surgery. Absence of the pituitary gland, but not the thyroid, adrenal or pineal glands, resulted in a significant decrease in tear SC, IgA and total protein content. In addition, removal of the thyroid or adrenal glands did not prevent the testosterone-associated increase in tear SC and IgA, although thyroidectomy or adrenalectomy did diminish the magnitude of the androgen response. In contrast, hypophysectomy completely blocked the effect of testosterone on both tear SC and IgA. These results indicate that the hypothalamic-pituitary axis may regulate, or mediate, the action of androgens on ocular immunity in the rat.     

Regulation of immune cell responses by semaphorins and their receptors

Semaphorins were originally identified as axon guidance factors involved in the development of the neuronal system. However, accumulating evidence indicates that several members of semaphorins, so-called ‘immune semaphorins'', are crucially involved in various phases of immune responses. These semaphorins regulate both immune cell interactions and immune cell trafficking during physiological and pathological immune responses. Here, we review the following two functional aspects of semaphorins and their receptors in immune responses: their functions in cell–cell interactions and their involvement in immune cell trafficking.     

Hormonal influence on the secretory immune system of the eye: androgen control of secretory component production by the rat exorbital gland

Androgens are known to regulate the level of secretory component (SC) in tears of male rats. The purpose of the present study was to explore the underlying mechanism of this hormone action by (i) identifying the ocular tissue(s) involved in SC production; and (ii) determining whether androgens increase SC production by this tissue. We also examined whether androgen administration influenced the concentration of SC in tears of female rats. Ocular tissues from adult Sprague-Dawley rats were cultured in the presence or absence of cycloheximide in the incubation medium. Secretory component in the culture media was measured by an RIA which detects primarily free SC. Analysis of media obtained after incubation of exorbital (lacrimal) glands, 'lid' tissues, globes, and Harderian glands revealed that only exorbital glands released substantial amounts of SC. This exorbital gland production of SC, which was significantly greater in tissues from male rats, as compared to those of female rats, was reduced by approximately 50% when cycloheximide was present in the culture medium. To determine whether SC production by exorbital glands was influenced by androgens, orchiectomized glands was influenced by androgens, orchiectomized rats were administered either saline or testosterone (2.0 mg/day for 4 days), and exorbital glands were cultured 24 hr after the last injection. Testosterone treatment in vivo induced a significant, cycloheximide-sensitive increase in SC production in vitro, compared to the glandular SC output of saline-injected controls. It is interesting that similar androgen treatment of ovariectomized females also resulted in elevated tear SC concentrations and enhanced output of SC by their exorbital glands in vitro. These findings indicate that the exorbital gland is primarily responsible for SC production in the rat eye and that androgens may modulate the synthesis of SC in this gland.     

Regulation of intestinal alkaline phosphatase levels in the rat: Role of the adrenal cortex

Bilateral adrenalectomy produces a reduction in the alkaline phosphatase concentration in rat intestine, which is not prevented by the administration of saline, but is prevented by the administration of cortisone. The administration of A.C.T.H. to normal rats leads to a rise in intestinal alkaline phosphatase levels associated with a small increase in the weight of the adrenals, while the administration of hydrocortisone produces large increases in the enzyme, even with doses which cause significant hypoplasia of the adrenal glands.The significance of these findings in relation to mechanisms of fat absorption and the treatment of steatorrhoea is discussed.     

Regulation of T cell responses by the receptor molecule Tim-3

Immunologic Research - Tim-3 is a member of the T cell immunoglobulin and mucin domain (Tim) family of proteins, which are expressed by several cell types in the immune system, including CD4 and...     

Possible autocrine regulation of chromaffin cell activity in adrenal glands of the frog by endothelin-1-induced serotonin release

The aim of the present study was the demonstration of mechanisms of regulation of activity of chromaffin cells in the adrenal gland of Rana ridibunda (Anura-Amphibia). Previous studies have shown that endothelin-1 is an important factor for the maintenance of adrenal gland function. On the basis of these findings, frogs were injected with [Ala(1,3,11,15)]-endothelin-1 (0.025 mg/0.2 ml), which is a selective agonist of the endothelin B receptor, whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 5, 20, and 60 min after injection and fixed, embedded in paraffin wax and stained by histological and immunohistochemical means, applied on adjacent 4-microm-thick sections. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase and serotonin 2A receptors were shown by the peroxidase-antiperoxidase (PAP)-3,3'-diaminobenzidine tetrachloride (DAB) method. After injection of [Ala(1,3,11,15)]-endothelin-1, chromaffin cells secreted serotonin and synthesized dopamine beta-hydroxylase. In conclusion, these findings suggest that [Ala(1,3,11,15)]-endothelin-1 stimulates chromaffin cell activity in frog adrenal glands. Moreover, the presence of serotonin 2A receptors in chromaffin cells indicates that these cells are also targets for serotonin and that there is an autocrine signaling pathway in chromaffin cells. This is the first report providing data on the effects of endothelin-1 on chromaffin cells in frog adrenal glands.