[3H]Spiroperidol binding in normal and denervated carotid bodies

Producción CientíficaSpecific dopamine receptors were studied in freshly dissected, unhomogenized rabbit carotid bodies incubated in [3H]spiroperidol. Total binding and non-specific binding were determined in the absence and presence of 0.2 #M (+)-butaclamol, respectively. Specific binding in normal carotid bodies incubated at near saturating concentrations (0.38 nM) was 1.63 _+ 0.58 pmol/g of tissue. Chronic section of the carotid sinus nerve (14 days) resulted in a 64070 reduction (P < 0.05) in specific binding. We conclude that the majority of specific dopaminergic receptors are located on carotid sinus nerve afferent terminals

Reciprocal modulation of tyrosine hydroxylasea activity in rat carotid body

The carotid body is an arterial chemoreceptor organ responsive to blood levels of pO2, pCOe and pH 13. The parenchymal tissue of the carotid body is composed mainly of two cell types: the glomus or Type I cells, which are disposed together in groups or glomeruli, and the sustentacular or Type II cells, which appear as glial-like elements enclosing the glomeruli in capsular fashion 3,4. The Type I cells, which have abundant dense-cored vesicles and are known to contain catecholaminesl, 2,11,15, receive a sensory innervation from afferent fibers of the carotid sinus nerve 3. Recent studies have also shown the presence of reciprocal synapses at these junctions between afferent nerve terminals and Type I cells TM. In addition, these cells receive an efferent innervation from both preganglionic and postganglionic sympathetic fibers which reach the carotid body from the superior cervical ganglion 1

Localization and Function of Cat Carotid Body Nicotinic Receptors

Producción CientíficaAcetylcholine and nicotinic agents excite cat carotid body chemoreceptors and modify their response to natural stimuli. The present experiments utilized [125I]a-bungarotoxin ([125I]a-BGT) to localize within the chemosensory tissue the possible sites of action of exogenous and endogenous nicotinic cholinergic substances. In vitro equilibrium binding studies of intact carotid bodies determined a K d of 5.57 nM and a Bma x of 9.21 pmol/g of tissue. Chronic section (12-15 days) of the carotid sinus nerve (CSN) did not change the amount of displaceable toxin binding. In contrast, the specific binding was reduced by 46% following removal of the superior cervical ganglion. Light microscope autoradiography of normal, CSN-denervated and sympathectomized carotid bodies revealed displaceable binding sites concentrated in lobules of type I and type II cells. Treatment of carotid bodies with 50 nM a-BGT in vitro reduced by 50% the release of [3H]dopamine (synthesized from [3H]tyrosine) caused by hypoxia or nicotine, and also significantly reduced the stimulus-. evoked discharges recorded from the CSN. The data suggest (1) an absence of ct-BGT binding sites on the afferent terminals of the CSN and (2) that nicotinic receptors located within parenchymal cell lobules may modulate the release of catecholamines from these cells

Alpha-bungarotoxin binding in cat carotid body

Producción CientíficaThe carotid body is an arterial chemosensory organ which detects changes in blood gas tensions and pH, and reflexly contributes to the cardiorespiratory adjustments which occur during hypoxia, hypercapnia and acidosis. However, the sensory mechanisms involved in carotid chemoreception remain to be elucidated. Morphologically, the carotid body consists of an association of elemental units, or glomeruli, within a connective tissue stroma penetrated by a dense capillary net 5. The glomeruli are comprised of catecholamine-rich type I, or chief cells, which are enveloped by glial-like processes of type II, or sustentacular, cellsa,4,19. Sensory fibers from the carotid sinus nerve penetrate the glomeruli to terminate in synaptic-like apposition on type I cellst,18, 21

Effects of low pH on synthesis and release of catecholamines in the cat carotid body in vitro

Producción CientíficaThe rates of dopamine and noradrenaline synthesis in the cat carotid body (c.b.) are 5.9 _+ 0.58 pmol/c.b./2 h and 0.3 + 0.02 pmol/c.b./2 h, respectively. The synthesis is doubled when the organs are incubated at pH 7. Similarly, low pH induces a release of dopamine from the c.b. which is proportional to increased activity in the carotid sinus nerve

Differential stimulus coupling to dopamine and norepinephrine stores in rabbit carotid body type I cells

Recent studies suggest that preneural type I (glomus) cells in the arterial chemoreceptor tissue of the carotid body act as primary transducer elements which respond to natural stimuli (low 02, pH or increased CO2) by releasing chemical transmitter agents capable of exciting the closely apposed afferent nerve terminals. These type I cells contain multiple putative transmitters, but the identity of the natural excitatory agents remains an unresolved problem in carotid body physiology. Characterization of putative transmitter involvement in the response to natural and pharmacological stimuli has therefore become fundamental to further understanding of chemotransmission in this organ. The present study demonstrates that a natural stimulus (hypoxia) evokes the release of dopamine (DA) and norepinephrine (NE) in approximate proportion to their unequal stores in rabbit carotid body (DA release/NE release = 8.2). In contrast, nicotine (100/~M), a cholinornimetic agent thought to act on the nicotinic receptors present on the type I cells, evokes the preferential release of NE (DA release/NE release = 0.17). These findings suggest that distinct mechanisms are involved in a differential mobilization of these two cateeholamines from the rabbit carotid body

Synthesis and Release of Catecholamines by the Cat Carotid Body in Vitro: Effects of Hypoxic Stimulation

Producción CientíficaThe role of catecholamines (CAs) in cat carotid body chemoreception has been controversial. On the basis of pharmacological experiments, it would appear that endogenous dopamine (DA) may act either as an inhibitory or excitatory transmitter. Neurochemical studies on the effects of natural stimulation on the release of carotid body CAs in the cat have also been inconclusive. In the present study, we have characterized the synthesis and release of CAs in the in vitro cat carotid body preparation in response to different levels of hypoxic stimulation and have correlated these measures with the chemosensory activity of the carotid sinus nerve. The synthesis of [3H]DA and [3H]norepinephrine was linear for at least 4 h in carotid bodies incubated with their natural precursor [~H]tyrosine. Synthesis of both [3H]CAs plateaued when the [3H]tyrosine concentration in the media reached 40 uM, which is a concentration similar to that found in cat plasma. Exposure of the animals to an atmosphere of 10% 02 in N~ for 3 h prior to removal and incubation of the carotid bodies with [3H]tyrosine resulted in an approximately 100% increase in the rate of [3H]DA synthesis but no change in [3H]norepinephrine synthesis. This selective increase in [3H]DA synthesis was not detected when [3H]dihydroxyphenylalanine was used as precursor. Carotid bodies first incubated with [3H]tyrosine and later superfused with solutions equilibrated with different gas mixtures (0 100% 0 2 in N2) exhibited an increase in [3H]DA release and carotid sinus nerve discharge which were inversely related to the oxygen concentration. This relationship was strongest for the weaker stimuli (between 50% and 20% O 2 in N2), where both nerve activity and [3H]DA release increased almost in parallel. With lower oxygen concentrations (10% O 2 and 0% 02 equilibrated solutions), the increase in the release of [3H]DA was proportionally greater than the increase in carotid sinus nerve discharge. Our results demonstrate that hypoxic stimulation increases both the rate of synthesis and release of DA in the cat carotid body. Although the precise role of DA in this chemoreceptor organ is presently unknown, our findings suggest that this biogenic amine plays a direct role in generating or controlling the electrical activity in the carotid sinus nerve. INTRODUCTION The mammalian carotid body is an arterial chemoreceptor organ activated by low paO2, low pH and high p~CO2 (ref. 21). Structurally, the receptor complex is formed by clusters of two types of cells, the type I and type II cells; the clusters lie within a supporting connective tissue matrix containing a dense capillary net24, 41. Sensory fibers of the carotid sinus nerve (CSN) penetrate these cell clusters to end in synaptic apposition with type I cells, which are considered to be preneural (receptor) elements. Two perennial issues pertaining to arterial chemoreception, concern first, whether the type 1 cells are in fac

A chronic pain: inflammation-dependent chemoreceptor adaptation in rat carotid body

Producción CientíficaExperiments in recent years have revealed labile electrophysiological and neurochemical phenotypes in primary afferent neurons exposed to specific stimulus conditions associated with the development of chronic pain. These studies collectively demonstrate that the mechanisms responsible for functional plasticity are primarily mediated by novel neuroimmune interactions involving circulating and resident immune cells and their secretory products, which together induce hyperexcitability in the primary sensory neurons. In another peripheral sensory modality, namely the arterial chemoreceptors, sustained stimulation in the form of chronic hypoxia (CH) elicits increased chemoafferent excitability from the mammalian carotid body. Previous studies which focused on functional changes in oxygen-sensitive type I cells in this organ have only partially elucidated the molecular and cellular mechanisms which initiate and control this adaptive response. Recent studies in our laboratory indicate a unique role for the immune system in regulating the chemo-adaptive response of the carotid body to physiologically relevant levels of hypoxia

Muscarinic receptor localization and function in rabbit carotid body

Producción CientíficaAcetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [SH]quinuclidinylbenzitate ([3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1/~M atropine) with a K d = 71.46 pM and a Bm~ x = 9.23 pmol/g tissue. These binding parameters and the pharmacology of the displaceable [SH]QNB binding sites are similar to specific muscannic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that musearinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [3H]QNB binding sites to Iobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100/~M) and bethanechol (100 ~tM) were shown to inhibit both the release of catecholamines and the increased CSN activity evoked by nicotine (50/~M) stimulation of the in vitro carotid body, Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites

Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Producción CientíficaCurrent concepts on the location and functional significance of nicotinic receptors in the carotid body rest on a-bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by prior incubation with the tritiated natural precursor w3Hxtyrosine, we have found that nicotine induces release of w3Hxcatecholamines in a dose-dependent manner IC50s9.81 mM.. We also found that mecamylamine 50 mM. completely abolished the nicotine-induced release, while a-bungarotoxin 100 nM; f20 times its binding Kd. only reduced the release by 56%. These findings indicate that chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to a-bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception