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 Dulbecco's 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-beta-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.

Neuiropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukmia cell intracellular calcium mobilization, perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.Neuropeptide Y (NPY) is a 36-amino acid peptide with an N-terminal tyrosine and a C-terminal tyrosine amide, first isolated from porcine brain by Tatemoto et al. in 1982 (1). NPY has been found to be an abundant mammalian neuropeptide, widely distributed throughout the central and peripheral nervous systems (2-4). On the basis of the pharmacological effects observed in experimental animals after central or peripheral administration of NPY, the peptide has tentatively been implicated in the regulation of a wide variety of biological functions such as vascular tone, feeding behavior, mood, and hormone secretion among others (for a review see ref. 5). At least two NPY receptor subtypes have been described based on the relative affinity of different NPY agonists: NPY-Y1 receptors require essentially the full NPY sequence of amino acids (see Fig. 1) for activation and have high affinity for the analog [Leu31,Pro34]NPY, whereas NPY-Y2 receptors can be activated by NPY and the shorter C-terminal fragment, NPY13-36, but have low affinity for [Leu31,Pro34]NPY (6,7). A third subtype (NPY-Y3) that recognizes all three of the above peptides but is insensitive to the NPY homolog, peptide YY, has been proposed (8, 9). Direct demonstration of a physiological and pathophysiological role for NPY has been hampered by the lack of specific, high-affinity NPY receptor antagonists. Receptor antagonists based on modified Cterminal fragments of NPY (10) Peptide Synthesis. Peptides were synthesized by the solidphase method. Compound 2 was obtained by oxidation of the reduced monomer and purification of the dimer by HPLC. Compound 3 was synthesized by using standard solid-phase synthesis. Compound 4 was synthesized by coupling BOC-Lglutamic acid fluorenylmethyl ester and a-Boc 3-FmOC-Ldiamino propionic acid in position 8 and 6, respectively. Dimerization was achieved on the resin by treatment with piperidine followed by a coupling reagent. Detailed synthesis is described in the compounds' patent publication (15).Binding Assays.[3H]NPY binding to rat brain membranes was done as described (16) except that incubations were terminated by filtration on a Brandel cell harvester through a Whatman GF/B filter, previously soaked overnight in 0.3% po...

Background Nearly all patients with newly diagnosed glioblastoma experience recurrence following standard-of-care radiotherapy (RT) + temozolomide (TMZ). The purpose of the phase 3 randomized CheckMate 548 study was to evaluate RT+TMZ combined with the immune checkpoint inhibitor nivolumab (NIVO) or placebo (PBO) in patients with newly diagnosed glioblastoma with methylated MGMT promoter (NCT02667587). Methods Patients (N=716) were randomized 1:1 to NIVO [(240 mg every 2 weeks ×8, then 480 mg every 4 weeks) + RT (60 Gy over 6 weeks) + TMZ (75 mg/m 2 once daily during RT, then 150-200 mg/m 2 once daily days 1-5 of every 28-day cycle ×6)] or PBO+RT+TMZ following the same regimen. The primary endpoints were progression-free survival (PFS) and overall survival (OS) in patients without baseline corticosteroids and in all randomized patients. Results As of December 22, 2020, median (m)PFS (blinded independent central review) was 10.6 months (95% CI, 8.9-11.8) with NIVO+RT+TMZ vs 10.3 months (95% CI, 9.7-12.5) with PBO+RT+TMZ (HR, 1.1; 95% CI, 0.9-1.3) and mOS was 28.9 months (95% CI, 24.4-31.6) vs 32.1 months (95% CI, 29.4-33.8), respectively (HR, 1.1; 95% CI, 0.9-1.3). In patients without baseline corticosteroids, mOS was 31.3 months (95% CI, 28.6-34.8) with NIVO+RT+TMZ vs 33.0 months (95% CI, 31.0-35.1) with PBO+RT+TMZ (HR, 1.1; 95% CI, 0.9-1.4). Grade 3/4 treatment-related adverse event rates were 52.4% vs 33.6%, respectively. Conclusions NIVO added to RT+TMZ did not improve survival in patients with newly diagnosed glioblastoma with methylated or indeterminate MGMT promoter. No new safety signals were observed.

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 or secretion induced by Ionomycin. The EC50 for nicotine is 3 microM for catecholamine secretion and 10 microM for 45Ca2+ uptake, while the EC50s for veratridine-stimulated uptake and secretion are approximately the same (75 microM). 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 50 mM-K+ show saturation kinetics with respect to external calcium concentrations at about 2 mM. On the other hand, the uptake of 45Ca2+ 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.

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 Locke's solution but has no effect on high concentrations of nicotine (20 microM). In Ca2+-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na+ with Li+ in Locke's 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. alpha-Bungarotoxin (1 microM) 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.

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Ornithine decarboxylase (ODC) and the polyamines are thought to play a role in maturation of mammalian tissues. Daily postnatal administration of alpha-difluoromethylornithine (DFMO, a specific inhibitor of ODC) to newborn rats caused organ-specific deficits in tissue weight gain, with brain and kidney as the major targets. Subnormal organ weights were associated with deficits in the levels of nucleic acids and proteins in the affected tissues, and examination of the synthetic rates of DNA ([3H]thymidine incorporation), RNA ([3H]uridine incorporation) and protein ([14C]leucine incorporation) confirmed that macromolecule synthesis was inhibited in DFMO-treated pups. The time of onset of effect of DFMO on the synthesis of nucleic acids and proteins was the same as that reported for depletion of polyamines by this treatment. Potential adverse effects of DFMO on cell survival were also assessed by labeling DNA with [3H]thymidine on day 3 and examining retention of label 12 days later; DFMO did not cause an increase in cell death. In contrast to the sensitivity of brain and kidney to postnatally administered DFMO, development of cardiac tissue was relatively resistant to growth inhibition despite polyamine depletion. The organ specificity of effect of DFMO results, in part, from the different timetables for cellular events in tissue development displayed by each organ type; administration of DFMO earlier in development (during days 15 to 17 of gestation) did produce deficiencies in cardiac growth and nucleic acid levels similar to those which had been seen for brain and kidney. These data support the view that polyamines play a key role in cell replication, differentiation and growth during critical periods of mammalian organ development through their regulation of DNA, RNA, and protein synthesis.

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 [(3)H]norepinephrine or [(3)H]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.