Adenosine Increases the cAMP Content of the Rat Carotid Body In Vitro

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice

AJP Cell Physiology ◽

10.1152/ajpcell.2002.282.1.c27 ◽

2002 ◽

Vol 282 (1) ◽

pp. C27-C33 ◽

Cited By ~ 58

Author(s):

L. He ◽

J. Chen ◽

B. Dinger ◽

K. Sanders ◽

K. Sundar ◽

...

Keyword(s):

Tyrosine Hydroxylase ◽

Nadph Oxidase ◽

Carotid Body ◽

Gene Knockout ◽

Type I ◽

Type I Cells ◽

Carotid Bodies ◽

Marker Antigen ◽

I Cells

Various heme-containing proteins have been proposed as primary molecular O2 sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91 phox ) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O2-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K+currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca2+ concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91 phox gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O2 sensor in arterial chemoreception.

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Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro

Journal of Applied Physiology ◽

10.1152/jappl.2000.88.4.1489 ◽

2000 ◽

Vol 88 (4) ◽

pp. 1489-1495 ◽

Cited By ~ 23

Author(s):

David F. Donnelly ◽

Ricardo Rigual

Keyword(s):

Mouse Model ◽

Carotid Body ◽

Unit Activity ◽

Action Potentials ◽

Single Unit ◽

Gene Deletions ◽

Suction Electrode ◽

Single Unit Recordings ◽

Unit Action

A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single-unit action potentials were 57 ± 10 (SE) ( n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate[Formula: see text] to near 20 Torr caused an increase in spiking activity from 1.3 ± 0.4 to 14.1 ± 1.9 Hz ( n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction.

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The Interaction Between Low Glucose and Hypoxia in the in vitro, Rat Carotid Body

Advances in Experimental Medicine and Biology - Arterial Chemoreception ◽

10.1007/978-94-007-4584-1_17 ◽

2012 ◽

pp. 123-127 ◽

Cited By ~ 3

Author(s):

Andrew P. S. Holmes ◽

David Hauton ◽

Prem Kumar

Keyword(s):

Carotid Body ◽

Low Glucose

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Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1982.243.2.g117 ◽

1982 ◽

Vol 243 (2) ◽

pp. G117-G126

Author(s):

R. Fogel ◽

G. W. Sharp ◽

M. Donowitz

Keyword(s):

Cholera Toxin ◽

Calcium Content ◽

Intracellular Camp ◽

Rabbit Ileum ◽

Camp Content ◽

Serosal Surface ◽

Ileal Absorption ◽

Camp Levels

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

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Effect of Na+ and K+ channel blockade on baseline and anoxia-induced catecholamine release from rat carotid body

Journal of Applied Physiology ◽

10.1152/jappl.1994.77.6.2606 ◽

1994 ◽

Vol 77 (6) ◽

pp. 2606-2611 ◽

Cited By ~ 25

Author(s):

T. P. Doyle ◽

D. F. Donnelly

Keyword(s):

Carotid Body ◽

K Channel ◽

Channel Blockers ◽

Nerve Activity ◽

Sham Treatment ◽

K Channels ◽

Nerve Response ◽

Carotid Bodies ◽

Carotid Body Glomus Cells

Ionic membrane currents are hypothesized to play a major role in determining secretion from carotid body glomus cells, and increased secretion likely mediates the increase in nerve activity in response to hypoxia. The hypothesis that Na+ and K+ channels play an important role in determining secretion and nerve activity was tested by measuring single-fiber afferent nerve activity along with an estimate of free tissue catecholamine using Nafion-covered carbon-fiber micro-electrodes placed in rat carotid bodies in vitro. Baseline and anoxia-stimulated (1 min duration; PO2 of approximately 0 Torr at nadir) levels were quantified. Sham treatment had no significant effect. Tetrodotoxin (2 microns) ablated the nerve activity and reduced peak catecholamine (19.5 +/- 3.1 to 14.5 +/- 3.4 microM; P < 0.05). Cesium (10 microns) had no effect on catecholamine but reduced the nerve response (19.8 +/- 2.7 to 7.8 +/- 2.0 Hz; P < 0.05). 4-Aminopyridine (4 mM) significantly reduced the nerve response (17.2 +/- 3.7 to 4.9 +/- 1.9 Hz; P < 0.05) and increased the baseline (0.9 +/- 0.2 to 3.1 +/- 0.8 microM; P < 0.05) and reduced the peak catecholamine (10.0 to 4.3 +/- 0.8 microM; P < 0.05) levels. These results demonstrate that Na+ and K+ channels play an important role in modulating the secretory and nerve responses. However, channel blockers do not emulate severe hypoxia, suggesting that hypoxia transduction procedes, at least in part, through an alternate pathway.

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Chemoreceptor nerve excitation may not be proportional to catecholamine secretion

Journal of Applied Physiology ◽

10.1152/jappl.1996.81.2.657 ◽

1996 ◽

Vol 81 (2) ◽

pp. 657-664 ◽

Cited By ~ 45

Author(s):

D. F. Donnelly

Keyword(s):

Carotid Body ◽

Catecholamine Secretion ◽

Nerve Activity ◽

Peripheral Chemoreceptor ◽

Carotid Bodies ◽

Carbon Fiber Microelectrodes ◽

S Peak ◽

Nerve Excitation ◽

Catecholamine Levels

Enhanced catecholamine secretion from the carotid body glomus cells is hypothesized to play an essential role in mediating the peripheral chemoreceptor response to hypoxia. To test aspects of this hypothesis, the relationship between catecholamine secretion and nerve activity was examined during repetitive hypoxia stimuli and after catecholamine depletion with reserpine. Single-fiber afferent serve activity was measured along with an estimate of free tissue catecholamine by using Nafion-coated carbon-fiber microelectrodes placed in rat carotid bodies in vitro. Baseline and stimulated nerve and catecholamine levels were quantified during repetitive stimulation (anoxia of 1-min duration; PO2 = 0 Torr at nadir, repeated each 200 s). Peak stimulated catecholamine progressively decreased from 26.4 +/- 2.6 microM for the first stimulus to 7.5 +/- 0.9 microM for the fifth stimulus (n = 15), but peak nerve activity was much less affected (23.0 +/- 1.9 Hz, first trial; 19.9 +/- 1.4 Hz, fifth trial). An exposure to moderate hypoxia (approximately 80 Torr) before the repetitive anoxia stimuli produced catecholamine levels comparable to those obtained during repetitive anoxia, but peak nerve activity was significantly less (22.5 +/- 3.4 vs. 12.7 +/- 2.1 Hz). Pretreatment with reserpine (1 mg/100 g) resulted in a large reduction in the average hypoxia-induced catecholamine response (1.4 +/- 0.3 microM, n = 9), but peak nerve activity was not different from nontreated controls. These results demonstrate an independence between carotid body catecholamine secretion and nerve activity, suggesting that nerve excitation is, at least, partially mediated through pathways independent of granule secretion.

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Reversible effects of phenothiazines on frog gastric stimulation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1984.247.4.g366 ◽

1984 ◽

Vol 247 (4) ◽

pp. G366-G376

Author(s):

N. Raphael ◽

E. B. Ekblad ◽

T. E. Machen

Keyword(s):

Adenylate Cyclase ◽

Time Course ◽

Phosphodiesterase Inhibitor ◽

Secretory Activity ◽

Gastric Stimulation ◽

Camp Content ◽

Oxyntic Cell ◽

Camp Generation ◽

Stimulus Secretion Coupling

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and promethazine (PZ) were tested for effects on stimulus-secretion coupling in in vitro bullfrog gastric mucosa. When added to histamine-stimulated tissues, the drugs caused H+ secretion to decrease and transepithelial resistance to increase over a 2-h time course. The potency sequence was TFP (IC50 = 40 microM) greater than CPZ (IC50 = 72 microM) congruent to PZ (IC50 = 72 microM). Anesthetics and other phenothiazines with weak anticalmodulin activity had no effect on secretory parameters. In the presence of histamine, further addition of isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) plus dibutyryl cAMP (DBcAMP), IBMX alone, or forskolin (a specific activator of adenylate cyclase) to phenothiazine-inhibited tissues caused full resumption of secretory activity. If TFP (50 microM) was added before stimulation with histamine, the normal increases in tissue cAMP content (which occurs primarily in oxyntic cells), oxyntic cell apical membrane elaboration (morphometric analysis of electron micrographs), and H+ secretion were all blocked. Subsequent addition of IBMX or IBMX plus DBcAMP completely reversed the TFP effect. These results indicate that the histamine-sensitive adenylate cyclase may be the site of TFP inhibition and Ca2+-calmodulin regulation; since these drugs inhibited stimulation by DBcAMP plus IBMX, they may also be exerting additional effects distal to cAMP generation.

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