Norman Kirshner

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.

The acetylcholine receptor of the adrenal medulla.

Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [1251]α‐bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α‐bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d‐tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α‐bungarotoxin.

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.

Endogenous inhibitor(s) in adrenal medulla of dopamine-ß-hydroxylase

Abstract The addition of Cu2+ ions to various subcellular fractions of the adrenal medulla caused marked increases in dopamine-s-hydroxylase activity. Further studies with Cu2+ and other sulfhydryl-reactive reagents such as Hg2+, Ag1+, p- hydroxymercuribenzoate , and N-ethylmaleimide indicate that the increased enzymatic activity was due to the inactivation of one or more endogenous sulfhydryl compounds. The addition of Ca2+, Mn2+, Zn2+, Fe2+, Fe3+, and Ni2+ to the various fractions of the medulla had no effect on dopamine-s-hydroxylase activity. The dopamine-s-hydroxylase activity in the subcellular fractions measured with and without the addition of Cu2+ confirms previous reports that the enzyme is largely localized in the catecholamine storage vesicles and also indicates that the enzyme is more easily solubilized than was previously thought.

Phosphorylation of Adrenal Medulla Cell Proteins in Conjunction with Stimulation of Catecholamine Secretion

Abstract: Enhanced phosphorylation of two specific protein bands accompanied catecholamine secretion from cultured bovine adrenal medulla cells stimulated by different secretagogues. Cells preincubated with 32Pi were treated with nicotine, veratridine, Ionomycin, or barium. Each of these secretagogues stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 95,000. Phosphorylation of the 60,000 M.W. protein band was two‐ to threefold higher than that of the 95,000 M.W. band on stimulation with nicotine, veratridine, or barium, but Ionomycin stimulated phosphorylation of each protein band to the same extent. In general, the increase in phosphorylation was most rapid during the first minute of stimulation and occurred prior to detectable secretion. Phosphorylation reached a relatively constant level within 5 min after onset of stimulation at a time when catecholamine release was still proceeding at a rapid rate. Nicotine‐stimulated phosphorylation and catecholamine secretion were calcium‐dependent and blocked by d‐tubocurarine, whereas tetrodotoxin inhibited veratridine‐stimulated secretion and phosphorylation. We conclude that catecholamine secretion and protein phosphorylation occur under similar conditions and that Ca2+‐dependent incorporation of phosphate into specific proteins may be a link in stimulus‐secretion coupling.

Release of Catecholamines and Specific Protein from Adrenal Glands

Rabbit antiserums directed against a purified protein obtained from the catecholamine storage vesicles of the adrenal medulla of the cow were used to study the mechanism of secretion from the adrenal medulla. During secretion evoked by acetylcholine in isolated perfused adrenal glands, catecholamines and the specific intravesicular protein were released in relative amounts comparable to those present in the intact gland. Thus catecholamines are apparently released from the storage vesicles by a process resembling reverse pinocytosis.

Biosynthesis of epinephrine and norepinephrine by sympathetic nerves and ganglia.

It is generally believed by the neurophysiologists that the sympathetic neurohormone is norepinephrine and not epinephrine as originally supposed. This concept of norepinephrine as the sympathetic neurohormone has evolved from much impressive circumstantial evidence; however, it has not been demonstrated that the biosynthesis of norepinephrine takes place in the sympathetic nerves and ganglia. This paper shows, through the use of labeled precursors, that the sympathetic nerves and ganglia synthesize norepinephrine in a systematic sequence from tyrosine and that this synthesis apparently terminates with norepinephrine rather than progressing to the formation of epinephrine.

Plasma Dopamine β-Hydroxylase: A Possible Aid in the Study and Evaluation of Hypertension

The activity of dopamine β-hydroxylase (DBH) in plasma ranged from 2 to 100 units per liter of plasma in 82 apparently healthy subjects (ages 22 to 35 years). A nonnormal pattern of distribution was evident: 62 subjects had values below 35 units (18 � 1), while 13 of the remaining 20 subjects had values above 60 units (80 � 5). Those with low DBH activity had lower values for urinary catecholamine excretion (31 � 3 micrograms), with normal and stable blood pressure; those with high DBH activity had higher values for urinary catecholamine excretion (72 � 6 micrograms), with greater lability of arterial blood pressure. The DBH activity was significantly elevated in patients with labile (74 � 2 mm-Hg) or fixed (57 � 2 mm-Hg) essential hypertension. The results indicate that plasma DBH activity is low and that it falls within a narrow range in young adults with normal and stable blood pressure.

Uptake of Catecholamines by a Particulate Fraction of the Adrenal Medulla

Chromaffin granules isolated from beef adrenal medulla took up C14-labeled catecholamines from an isotonic medium at 37�C at a rate approximately 20 times the rate at 0�C. The uptake was stimulated three- to five-fold by Mg++ and adenosine triphosphate. Reserpine (1 x 10-5M) caused a 90 percent inhibition of uptake; ethylenediaminetetraacetic acid completely inhibited the stimulation by magnesium and adenosine triphosphate, and the inhibition could be reversed by addition of excess Mg++.