Ruta Slepetis

Stability of Bovine Adrenal Medulla Cells in Culture

: The functional stability of primary cultures of adrenal medulla cells was investigated. Isolated cells were prepared by treatment of bovine adrenal glands with collagenase followed by purification on Percoll density gradients and were maintained in Dulbeccos medium containing 10% fetal calf serum. Within 12 h after plating on plastic culture dishes, the cells became firmly attached and exhibited good survival for periods of time up to 3 weeks, as indicated by their morphology using light and electron microscopy, by maintenance of their content of catecholamines, tyrosine hydroxylase, dopamine‐β‐hydroxylase, and phenylethanolamine N‐methyltransferase, and their ability to respond to secretagogues. During the first 10 days to 2 weeks in culture there was little or no change in any of these parameters. During the 3rd week there were progressive losses of catecholamine and enzyme activities and increased vacuolization of medullary cells. The cells synthesized protein and RNA with no apparent loss in activities over the period studied, but did not incorporate [3H]thymidine into PCA‐precipitable material. The cells responded to secretagogues and secretory antagonists similarly to isolated perfused adrenal glands. The studies described here demonstrate that primary cultures of adrenal medulla cells provide an excellent experimental system for obtaining more detailed information on stimulus‐secretion coupling and other functional aspects of the adrenal medulla.

Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells.

Abstract: The uptake of 45Ca2+ and secretion of catecholamines by primary cultures of adrenal medulla cells were studied. Nicotine, veratridine, potassium, and Ionomycin stimulate both the accumulation of 45Ca2+ and the secretion of catecholamines. Nicotinic antagonists block 45Ca2+ uptake induced by nicotine, tetrodotoxin blocks 45Ca2+ uptake induced by veratridine, and D600 blocks uptake induced by K+, nicotine, and veratridine, but not 45Ca2+ uptake or secretion induced by Ionomycin. The EC50 for nicotine is 3 μm for catecholamine secretion and 10 μm for 45Ca2+ uptake, while the EC50S for veratridinestimulated uptake and secretion are approximately the same (75 μm). Kinetic studies show that the uptake of Ca2+ is rapid and appears to precede the secretion of catecholamines, and that the rate of uptake declines rapidly. The uptake of 45Ca2+ and secretion of catecholamines stimulated by veratridine and 50 mm‐K+ show saturation kinetics with respect to external calcium concentrations at about 2 mm. On the other hand, the uptake of 45 Ca2+ stimulated by nicotine does not become saturated at external calcium concentrations of 10 mm although the secretion of catecholamines reaches a maximum at external calcium concentrations of 2 mm. The data suggest that depolarizing agents such as veratridine and 50 mm‐K+ stimulate 45Ca2+ entry through voltage‐sensitive calcium channels, while nicotinic agonists stimulate calcium entry through the acetylcholine receptor ion channels as well as through voltage‐sensitive calcium channels.

Ion Channels and Membrane Potential in Stimulus-Secretion Coupling in Adrenal Medulla Cells

Abstract: The role of Na+ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)‐sensitive Na+ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Lockes solution but has no effect on high concentrations of nicotine (20 μM). In Ca2+‐sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na+ with Li+ in Lockes solution reduces the response to nicotine and to veratridine. Complete replacement of Na+ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM‐100 mM‐K+, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM‐K+. Neither dibutyryl‐cyclic AMP nor dibutyryl‐cyclic GMP evokes secretion. α‐Bungarotoxin (1 μM) did not inhibit nicotine‐induced secretion. These studies indicate that Na+ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca2+. The membrane potential appears to affect nicotine‐ and veratridine‐evoked secretion.

Critical periods for the role of ornithine decarboxylase and the polyamines in growth and development of the rat: Effects of exposure to α-difluoromethylornithine during discrete prenatal or postnatal intervals.

The roles of ornithine decarboxylase (ODC) and the polyamines in fetal and neonatal development were examined through the use of α‐difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Administration to pregnant rats of 500 mg/kg of DFMO every 12 h for a 4‐day period (8 DFMO injections) resulted in fetal and neonatal death; DFMO early in gestation produced fetal resorption whereas late gestational exposure did not compromise fetal viability but instead resulted in a delayed toxic effect, with high mortality in the first postnatal week. Generalized toxicity of DFMO was not apparent in later developmental periods, as 4 days of DFMO treatment begun postnatally did not produce any neonatal death. Shortening the course of gestational DFMO treatment to 2.5 days (5 DFMO injections) also did not adversely affect fetal or neonatal viability and thus permitted identification of critical periods in which various tissues are sensitive to DFMO. Examination of growth patterns of brain, heart and kidney and of neurochemical development of central and peripheral catecholaminergic neurons indicated that different critical periods exist for effects of DFMO on each tissue or even on the various cell types within a tissue. The separable sensitivities were apparent even though the effects of DFMO on ODC and the polyamines for any given treatment period were fairly uniform in all tissues studied. These results indicate that the ODC/polyamine system plays multiple roles in fetal survival and in tissue growth during discrete periods of development; because the time course of cellular maturation differs for each tissue or cell population, DFMO administered during any one brief period can produce organ‐specific developmental deficits.

Inhibition of Catecholamine Secretion from Adrenal Medulla Cells by Neurotoxins and Cholinergic Antagonists

Abstract: The effects of several neurotoxins and cholinergic antagonists on the nicotine‐induced secretion of catecholamines by adrenal medulla cells in culture were investigated. Aconitine, veratridine, and batrachotoxin, in the presence of 1 μm‐tetrodotoxin inhibited the nicotine‐stimulated secretion of catecholamines in a dose‐dependent manner in Lockes solution. In Na+‐free sucrose medium, tetrodotoxin was not required to inhibit the stimulatory effects of aconitine, veratridine, and batrachotoxin, and these agents by themselves inhibited the nicotine‐stimulated secretion of catecholamines. Scorpion venom, which also increases the flux of Na+ through tetrodotoxin‐sensitive channels, was not an effective inhibitor of nicotine‐stimulated secretion. Histrionicotoxin, atropine, hexamethonium, and decamethoniun–as well as the Na+‐channel activators–noncompetitively inhibit nicotine‐stimulated secretion. The effects of these agents on nicotine‐stimulated secretion appear similar to their effects on the inhibition of depolarization at the neuromuscular junction. Reversibility studies suggest that the stimulatory and inhibitory sites of the neurotoxins are different, while studies in Na+‐free media suggest that tetrodotoxin‐insensitive sodium channels are not involved in the inhibitory effect of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins is the nicotinic‐receptor‐associated ion channel.

Role of ornithine decarboxylase and the polyamines in nervous system development: Short-term postnatal administration of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase.

The role of ornithine decarboxylase (ODC) and the polyamines in development of central and peripheral catecholaminergic neurons was examined through the use of α‐difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Short‐term postnatal administration of DFMO (500 mg/kg daily on days 1–6) to neonatal rats resulted in effective inhibition of ODC and depletion of both putrescine and spermidine in brain, heart and kidney; after cessation of DFMO administration, polyamine levels returned to normal by 10–13 days of age. There were no signs of generalized toxicity of short‐term DFMO treatment, as body weight gains were largely unaffected over the course of study (through weaning). However, development of peripheral sympathetic neurons was severely retarded by DFMO, with persistent and profound deficits of both cardiac and renal norepinephrine; the catecholamine deficiencies were unrelated to effects on end‐organ growth, as cardiac weights were essentially normal whereas kidney weights were adversely affected by DFMO. Development of the adrenal medulla, a peripheral catecholaminergic tissue which displays approximately the same developmental profile as do sympathetic neurons but which does not develop axonal projections, was not slowed by DFMO treatment; similarly, central noradrenergic and dopaminergic neurons, which undergo the majority of axonal outgrowth and synaptogenesis during the second to third postnatal week (just after the period in which polyamines returned to control levels), developed normally as assessed by measurements of transmitter levels, tyrosine hydroxylase activity and synaptosomal uptake of [3H]norepinephrine or [3H]dopamine. Extension of the period of DFMO treatment and consequent depletion of polyamines into the period in which central synaptogenesis occurs does, however, produce slowing of development of brain catecholamine neuronal projections. Thus, the ODC/polyamine system appears to play a critical postnatal role in catecholamine systems specifically undergoing active synaptogenesis.

Differential secretion of opioid peptides and catecholamines from cultured cells of a human pheochromocytoma tumor

Abstract Dissociated cells from a human pheochromocytoma tumor were maintained in culture, and the secretion of opioid peptides (OP), endogenous catecholamines (CA) and preloaded [3H] norepinephrine from these cells was examined. Nicotine, veratridine, barium or Ionomycin stimulated the secretion of OP, endogenous CA and 3H from the pheochromocytoma cells. In general, the different secretagogues were more potent in releasing OP than endogenous CA; 3H secretion was intermediate. Secretion of OP was more sensitive to stimulation by the calcium ionophore Ionomycin and by veratridine than was CA secretion. Nicotine-evoked OP secretion was more sensitive to extracellular calcium concentration than was secretion of CA or 3H. In contrast, bovine adrenal chromaffin cells show no such differential secretion of OP and CA in response to Ionomycin stimulation or to nicotine stimulation under conditions of varying extracellular calcium concentration. The results show that human pheochromocytomas secrete OP as well as CA and that there may be heterogeneous storage pools of CA and OP in cultured pheochromocytoma cells.

Inhibition of 45Ca2+ uptake and catecholamine secretion by phenothiazines and pimozide in adrenal medulla cell cultures

The inhibition by several phenothiazine drugs and pimozide of the uptake of 45Ca2+ and secretion of catecholamines by cultured adrenal medulla cells stimulated with nicotine, veratridine, 50 mM K+, ionomycin and Ba2+ was studied. The inhibition of 45Ca2+ uptake, except for ionomycin, closely parallelled the inhibition of catecholamine secretion. The nicotine-and veratridine-stimulated effects were several fold more sensitive to inhibition by the drugs than were those stimulated by 50mM K+, ionomycin and Ba2+; the ionomycin-stimulated effects were least sensitive to inhibition. These studies indicate that the drugs have multiple effects on stimulus-secretion coupling in adrenal medulla cells. It is suggested that inhibition of the veratridine- and nicotine-stimulated events is due to membrane perturbations caused by the drugs, that inhibition of the 50mM K+- and Ba2+-stimulated events is due to alterations in the voltage sensitive membrane Ca2+ channel, and that inhibition of secretion elicited by ionomycin may be due to inhibition of Ca2+-calmodulin reactions or to more profound non specific membrane effects.

Effects of α-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase, on development of brain histamine and catecholamine systems in the neonatal rat

Daily administration of FMH to neonatal rats produced long-lasting inhibition of histidine decarboxylase in hypothalamus and cerebral cortex and led to depletion of histamine in both brain regions. The onset of depletion was more rapid in cerebral cortex, a region in which non-neurotransmitter pools of histamine predominate in early postnatal life, appearing as early as postnatal day 3; depletion in the hypothalamus, a region rich in histaminergic neuronal projections, appeared later. No effects were seen on body or brain growth, nor was development of other biogenic amine systems affected. FMH thus provides a selective probe for examining the role of histamine in brain development.

Ornithine decarboxylase and polyamines in developing rat brain and heart: Effects of perinatal hypothyroidism.

Hypothyroidism induced by perinatal administration of propylthiouracil (PTU) had profound effects on growth of the heart, with major organ weight deficits persisting well beyond the termination of drug treatment. These effects were preceded by disruption of the developmental patterns of cardiac ornithine decarboxylase (ODC) and the polyamines, which are thought to be intracellular modulators of cellular maturation. Activity of cardiac ODC was depressed in the PTU‐treated group and putrescine and spermidine levels were markedly subnormal. PTU administration also affected brain growth, but much less so than in the heart. The disruption of the brain ODC/polyamine system was also less pronounced, with relatively small degrees of spermidine depletion and a slight elevation of ODC. For both tissues, the biochemical effects of perinatal hypothyroidism were opposite to those found previously for administration of exogenous thyroid hormones. These results support the views that: (1) endogenous thyroid hormones contribute to the maintenance of normal developmental patterns of ODC and the polyamines, and (2) the ODC/polyamine system participates in events modulating subsequent tissue growth.