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J C Brooks

Publications and source records attributed to J C Brooks.

At least 37 records · Page 2Linked to original sources
PubMed1987

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells.

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells.

Permeabilized cells treated with the adenosine triphosphate analog, [35S]adenosine-5'-0-(3-thiotriphosphate) ([gamma-35S]ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35S from the cells over a 20 min period. Treatment of permeabilized cells with ATP gamma S inhibited secretion and 35S incorporation into the cells. Pretreatment with ATP gamma S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35S from [gamma-35S]ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells.

Adenosine Triphosphate↗
PubMed1984

Thiophosphorylation prevents catecholamine secretion by chemically skinned chromaffin cells.

Skinned cells treated with the adenosine triphosphate analog, adenosine-5'-0-(3-thiotriphosphate) showed calcium-dependent thiophosphorylation of cellular constituents. Catecholamine secretion was inhibited when the analog was used as the substrate to promote secretion. The attenuation of secretion was proportional to the percentage of the analog in mixtures with adenosine triphosphate. Moreover, cells treated with the analog were subsequently unable to secrete when presented with MgATP, their normal substrate, indicating that the secretory systems was locked in the thiophosphorylated state. We hypothesize that phosphorylation is the calcium-dependent step required to prime the secretory system for secretion while dephosphorylation is the event required for exocytosis.

Adenosine Triphosphate↗
PubMed1984

Effect of trifluoperazine and calmodulin on catecholamine secretion by saponin-skinned cultured chromaffin cells.

We have examined the effect of trifluoperazine on catecholamine secretion by chemically skinned, cultured adrenal chromaffin cells. These cells require only ATP and calcium for secretion. Catecholamine secretion was unaffected by the drug in the presence or absence of calcium and ATP over the range 0.1 to 10 microM. At 100 microM trifluoperazine, catecholamine release was calcium and ATP independent and represented 70-80% of the total cellular content. High concentrations of exogenous calmodulin had no effect on secretion in the presence or absence of calcium. We conclude that low concentrations of the drug have no effect on secretion, while high concentrations cause non-physiological catecholamine release.

Adenosine Triphosphate↗
PubMed1983

Cobaltous chloride-induced hypothermia in mice III: effect of pretreatment with 5-hydroxytryptaminergic agents.

The influence of various 5-hydroxytryptaminergic agonist and antagonist drugs on body-temperature response to cobaltous chloride in mice was noted. Pretreatment with p-chloroamphetamine, p-chlorophenylalanine, and p-iodoamphetamine antagonized the body-temperature response to cobalt. p-Chloroamphetamine and p-chlorophenylalanine reduced, while p-iodoamphetamine elevated, brain serotonin levels. The uptake inhibitor agents, fluoxetine and nisoxetine, failed to modify the ability of p-chloroamphetamine to antagonize cobalt hypothermia. Cyproheptadine, methergoline, and xylamidine pretreatment enhanced rather than antagonized body-temperature depression by cobalt. Tryptophan hydroxylase inhibitors antagonized cobalt hypothermia, but receptor-blocking agents were without influence, indicating that antagonism was mediated through mechanisms other than the depletion of serotonin. Elevation rather than depletion of brain serotonin by p-iodoamphetamine and failure of uptake inhibitors to modify p-chloroamphetamine antagonism of cobalt hypothermia lend further support for a nonserotonergic role of these amines in their ability to antagonize body-temperature depression by cobaltous chloride in mice.

Animals↗
PubMed1983

Catecholamine secretion by chemically skinned cultured chromaffin cells.

The secretory system of intact chromaffin cells is not accessible to direct chemical manipulation because of the selective permeability of the plasmalemma. We have devised a simple procedure for chemically "skinning" (permeabilizing) cultured adrenal medullary chromaffin cells by brief exposure to the detergent saponin. This procedure disrupts the continuity of the plasmalemma, thus allowing us to bypass those aspects of the secretory process controlled by the cell membrane and giving direct access to exogenous substances to the cellular secretory machinery. We report here that the skinned cells retain a fully competent secretory mechanism dependent only on exogenous calcium and MgATP. Saponin treatment had no significant effect on the total catecholamine content of the cells. Secretion could be initiated by either MgATP or calcium as long as the other was present in the medium. Catecholamine and dopamine-beta-hydroxylase release by the skinned cells was dependent on the calcium concentration of the medium. The ratio of secreted catecholamine and enzyme was similar to that of the cells, indicating that secretion occurred by an exocytotic mechanism. About half the total cellular content of the cytoplasmic enzyme lactic dehydrogenase was released during the permeabilization process and subsequent incubations, indicating plasmalemma permeability to molecules as large as protein. Calcium-induced secretion was unaffected by several drugs known to affect catecholamines and granule function. Saponin treatment of chromaffin cells in culture appears to be a simple means for allowing access to exogenous substances to the cells' secretory machinery. Therefore, it offers the opportunity to use chemical treatments, and perhaps specific antibodies to cellular components, to determine the role of these elements in the secretory process. These techniques should also be applicable to other cells known to secrete by an exocytotic mechanism.

Adenosine Triphosphate↗
PubMed1981

Effect of various stimulus parameters on electrically-induced catecholamine secretion by thin slices of bovine adrenal medulla.

We examined the role of various stimulus parameters in electrically-induced catecholamine secretion by thin slices of bovine adrenal medulla. The stimulus parameters examined were voltage, duration, pulse width, and frequency for square-wave monophasic pulses. As each was examined it was held constant at a selected value for the evaluation of subsequent stimulus characteristics. For 16 mm2 tissue slices, catecholamine secretion was approximately linearly related to stimulus voltage over the range 20--80 volts, with a threshold of 20 V. Increasing the voltage beyond 80 V did not enhance secretion. Similarly, catecholamine secretion was dependent upon the frequency of stimulation. For stimuli delivered at 50 V for a 10-sec interval there was a four-fold increase in secretion over the frequency range 10--100/sec. Increasing pulse width at a constant voltage (50 V) over the range 0.4--2.0 msec resulted in a four-fold increase in catecholamine secretion. For pulses of 50 V, 50/sec and 0.8 msec pulse width, secretion was dependent upon the duration of the stimulus. Enhanced secretion was evident for times as short as 2 sec; between 5 and 15 sec of stimulation catecholamine secretion was linearly related to stimulus duration. Over the range 2--15 sec there was a five-fold enhancement of secretion. Electrically-induced catecholamine secretion by slices was markedly dependent upon stimulus parameters. In general, it was enhanced by increasing voltage, stimulus duration, pulse width and frequency. For most experiments a good choice of stimulus parameters appears to be 50 V 10 sec duration, 0.8 msec pulse width delivered at a frequency of 50/sec. Maximizing all stimulus parameters resulted in a 17-fold enhancement of secretion.

Adrenal Medulla↗
PubMed1979

p-Chloroamphetamine antagonism of cobaltous chloride-induced hypothermia in mice.

Serotonin levels were measured in the brains of mice at 2 h and 10 days following the administration of a single dose of 10 mg/kg, i.p. of p-chloroamphetamine HCl (PCA). A 44% reduction in the concentration of brain serotonin was noted 2 h after PCA injection. No significant change in brain serotonin was observed 10 days following PCA administration. The time course of the body temperature response to cobaltous chloride (25 mg/kg, i.p.) was recorded 60 min following pretreatment with PCA (10 mg/kg, i.p.). PCA reduced the hypothermic response by approximately 60%, presumably through the depletion of central 5-hydroxytryptamine.

Amphetamines↗
PubMed1979

Cobaltous chloride-induced hypothermia. II: Pretreatment with sympathoplegics, antihistamines, and narcotic antagonists.

Body temperature depression was noted in rats, mice, and hamsters following intraperitoneal cobaltous chloride administration (25 mg/kg). Intracerebral cobalt injection elicited hypotermia in rats and mice but not in hamsters. Body temperature depression appeared to be centrally mediated in rats and mice and peripherally mediated in hamsters. The effect of intraperitoneal and intracerebral pretreatment with phentolamine, diphenhydramine, propranolol, cimetidine, and naloxone on the mouse rectal temperature response to cobalt (25 mg/kg ip) was noted. Systemic phentolamine injection (intraperitoneal) did not alter the cobalt response, whereas intracerebral administration partially antagonized cobalt-induced hypothermia, indicating that antagonism was mediated centrally. Pretreatment with propranolol and cimetidine failed to modify the temperature response. Intracerebral diphenhydramine did not influence cobalt hypothermia. However, this agent reduced the cobalt response when given intraperitoneally, presumably through a peripheral inhibitory mechanism. The intracerebral injection of naloxone 30 min prior to cobalt slightly enhanced hypothermia, apparently through a central action. Intracerebral 6-hydroxydopamine injection depleted brain norepinephrine and dopamine but exhibited no apparent influence on cobalt-induced hypothermia.

Animals↗