Biomedical subjects

N R Prabhakar

Publications and source records attributed to N R Prabhakar.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cellular mechanisms of oxygen sensing at the carotid body: heme proteins and ion channels.

The purpose of this article is to highlight some recent concepts on oxygen sensing mechanisms at the carotid body chemoreceptors. Most available evidence suggests that glomus (type I) cells are the initial site of transduction and they release transmitters in response to hypoxia, which in turn depolarize the nearby afferent nerve ending, leading to an increase in sensory discharge. Two main hypotheses have been advanced to explain the initiation of the transduction process that triggers transmitter release. One hypothesis assumes that a biochemical event associated with a heme protein triggers the transduction cascade. Supporting this idea it has been shown that hypoxia affects mitochondrial cytochromes. In addition, there is a body of evidence implicating non-mitochondrial enzymes such as NADPH oxidases, NO synthases and heme oxygenases located in glomus cells. These proteins could contribute to transduction via generation of reactive oxygen species, nitric oxide and/or carbon monoxide. The other hypothesis suggests that a K(+) channel protein is the oxygen sensor and inhibition of this channel and the ensuing depolarization is the initial event in transduction. Several oxygen sensitive K(+) channels have been identified. However, their roles in initiation of the transduction cascade and/or cell excitability are unclear. In addition, recent studies indicate that molecular oxygen and a variety of neurotransmitters may also modulate Ca(2+) channels. Most importantly, it is possible that the carotid body response to oxygen requires multiple sensors, and they work together to shape the overall sensory response of the carotid body over a wide range of arterial oxygen tensions.

Animals↗

PubMed2000

Involvement of substance P in neutral endopeptidase modulation of carotid body sensory responses to hypoxia.

Previously, we showed that carotid bodies express neutral endopeptidase (NEP)-like enzyme activity and that phosphoramidon, a potent inhibitor of NEP, potentiates the chemosensory response of the carotid body to hypoxia in vivo. NEP has been shown to hydrolyze methionine enkephalin (Met-Enk) and substance P (SP) in neuronal tissues. The purpose of the present study is to determine whether NEP hydrolyzes Met-Enk and SP in the carotid body and if so whether these peptides contribute to phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anesthetized adult cats (n = 72 carotid bodies). The hydrolysis of Met-Enk and SP was analyzed by HPLC. The results showed that both SP and Met-Enk were hydrolyzed by the carotid body, but the rate of Met-Enk hydrolysis was approximately fourfold higher than that of SP. Phosphoramidon (400 microM) markedly inhibited SP hydrolysis ( approximately 90%) but had only a marginal effect on Met-Enk hydrolysis ( approximately 15% inhibition). Hypoxia (PO(2), 68 +/- 6 Torr) as well as exogenous administration of SP (10 and 20 nmol) increased the sensory discharge of the carotid body in vitro. Sensory responses to hypoxia and SP (10 nmol) were potentiated by approximately 80 and approximately 275%, respectively (P < 0.01), in the presence of phosphoramidon. SP-receptor antagonists Spantide (peptidyl) and CP-96345 (nonpeptidyl) either abolished or markedly attenuated the phosphoramidon-induced potentiation of the sensory response of the carotid body to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and that SP is involved in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Animals↗

PubMed2000

Blunted respiratory responses to hypoxia in mutant mice deficient in nitric oxide synthase-3.

In the present study, the role of nitric oxide (NO) generated by endothelial nitric oxide synthase (NOS-3) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-3. Experiments were performed on awake and anesthetized mutant and wild-type (WT) control mice. Respiratory responses to 100, 21, and 12% O(2) and 3 and 5% CO(2)-balance O(2) were analyzed. In awake animals, respiration was monitored by body plethysmography along with O(2) consumption (VO(2)) and CO(2) production (VCO(2)). In anesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Under both experimental conditions, WT mice responded with greater increases in respiration during 12% O(2) than mutant mice. Respiratory responses to hyperoxic hypercapnia were comparable between both groups of mice. Arterial blood gases, changes in blood pressure, VO(2), and VCO(2) during hypoxia were comparable between both groups of mice. Respiratory responses to cyanide and brief hyperoxia were attenuated in mutant compared with WT mice, indicating reduced peripheral chemoreceptor sensitivity. cGMP levels in the brain stem during 12% O(2), taken as an index of NO production, were greater in mutant compared with WT mice. These observations demonstrate that NOS-3 mutant mice exhibit selective blunting of the respiratory responses to hypoxia but not to hypercapnia, which in part is due to reduced peripheral chemosensitivity. These results support the idea that NO generated by NOS-3 is an important physiological modulator of respiration during hypoxia.

Animals↗

PubMed2000

L-type Ca(2+) channel activation regulates induction of c-fos transcription by hypoxia.

In the present study we examined the intracellular pathways that link hypoxia to activation of c-fos gene expression. Experiments were performed on rat pheocromocytoma-12 (PC-12) cells. c-fos mRNA and promoter activities were analyzed by RT-PCR and reporter gene assays, respectively. BAPTA, a Ca(2+) chelator, inhibited c-fos mRNA and promoter activation by hypoxia. Nitrendipine, an L-type Ca(2+)-channel blocker, abolished, whereas BAY K 8644, an L-type channel agonist, enhanced c-fos activation by hypoxia. Ca(2+) currents were augmented reversibly by hypoxia, suggesting that Ca(2+) influx mediated by L-type Ca(2+) channels is essential for c-fos activation by hypoxia. We next determined downstream pathways activated by intracellular Ca(2+) concentration. Immunoblot analysis revealed Ca(2+)/calmodulin-dependent kinase II (CaMKII) protein in PC-12 cells and revealed that hypoxia increased the enzyme activity. KN-93, a CaMK inhibitor, blocked CaMKII activation and c-fos promoter stimulation by hypoxia. Ectopic expression of an active mutant of CaMKII (pCaMKII290) stimulated c-fos promoter activity under normoxia. Hypoxia increased phosphorylation of CREB at the serine residue 133 (Ser-133), and KN-93 attenuated this effect. Point mutations at the Ca(2+)/cAMP-responsive cis-element (Ca/CRE) attenuated, whereas point mutations in the serum-responsive cis-element (SRE) abolished transcriptional activation of c-fos by hypoxia. These results demonstrate that c-fos activation by hypoxia involves CaMK activation and CREB phosphorylation at Ser-133 and requires Ca/CRE and SRE. These observations demonstrate that Ca(2+)-dependent signaling pathways play a crucial role in induction of c-fos gene expression, which may underlie long-term adaptive responses to hypoxia.

Animals↗

PubMed2000

Oxygen sensing by the carotid body chemoreceptors.

Carotid bodies are sensory organs that detect changes in arterial blood oxygen, and the ensuing reflexes are critical for maintaining homeostasis during hypoxemia. During the past decade, tremendous progress has been made toward understanding the cellular mechanisms underlying oxygen sensing at the carotid body. The purpose of this minireview is to highlight some recent concepts on sensory transduction and transmission at the carotid body. A bulk of evidence suggests that glomus (type I) cells are the initial site of transduction and that they release transmitters in response to hypoxia, which causes depolarization of nearby afferent nerve endings, leading to an increase in sensory discharge. There are two main hypotheses to explain the transduction process that triggers transmitter release. One hypothesis assumes that a biochemical event associated with a heme protein triggers the transduction cascade. The other hypothesis suggests that a K(+) channel protein is the oxygen sensor and that inhibition of this channel by hypoxia leading to depolarization is a seminal event in transduction. Although there is body of evidence supporting and questioning each of these, this review will try to point out that the truth lies somewhere in an interrelation between the two. Several transmitters have been identified in glomus cells, and they are released in response to hypoxia. However, their precise roles in sensory transmission remain uncertain. It is hoped that future studies involving transgenic animals with targeted disruption of genes encoding transmitters and their receptors may resolve some of the key issues surrounding the sensory transmission at the carotid body. Further studies are necessary to identify whether a single sensor or multiple oxygen sensors are needed for the transduction process.

Animals↗

PubMed2000

HERG-Like potassium current regulates the resting membrane potential in glomus cells of the rabbit carotid body.

Direct evidence for a specific K(+) channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go-related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K(+) current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the resting membrane potential. Raising extracellular K(+) revealed a slowly deactivating inward tail current indicative of HERG-like K(+) current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC(50) = 13 +/- 4 nM, mean +/- SE) and high concentrations of Ba(2+) (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current (V(h) = -44 +/- 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, - 48 +/- 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca(2+)](i) in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.

Animals↗

PubMed2000

Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.

Previous studies have suggested that voltage-gated Ca(2+) influx in glomus cells plays a critical role in sensory transduction at the carotid body chemoreceptors. The purpose of the present study was to determine the effects of hypoxia on the Ca(2+) current in glomus cells and to elucidate the underlying mechanism(s). Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca(2+) current was monitored using the whole cell configuration of the patch-clamp technique, with Ba(2+) as the charge carrier. Hypoxia (pO(2) = 40 mmHg) augmented the Ca(2+) current by 24 +/- 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in a CO(2)/HCO(3)(-)-, but not in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was lowered from 7.4 to 7. 0, hypoxia augmented the Ca(2+) current by 20 +/- 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca(2+) channel blocker (2 microM, n = 6), prevented, whereas, omega-conotoxin MVIIC (2 microM, n = 6), an inhibitor of N and P/Q type Ca(2+) channels, did not prevent augmentation of the Ca(2+) current by hypoxia, implying that low oxygen affects L-type Ca(2+) channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide (2 microM, n = 8, at 0 mV), prevented, whereas, a protein kinase A inhibitor (4 nM PKAi, n = 10) did not prevent the hypoxia-induced increase of the Ca(2+) current. Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator, augmented the Ca(2+) current by 20 +/- 3% (n = 8, at 0 mV). In glomus cells treated with PMA overnight (100 nM), hypoxia did not augment the Ca(2+) current (-3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKCdelta-like immunoreactivity in the cytosol of the glomus cells. Following hypoxia (6% O(2) for 5 min), PKCdelta-like immunoreactivity translocated to the plasma membrane in 87 +/- 3% of the cells, indicating PKC activation. These results demonstrate that hypoxia augments Ca(2+) current through L-type Ca(2+) channels via a PKC-sensitive mechanism.

Animals↗

PubMed2000

Role of substance P in neutral endopeptidase modulation of hypoxic response of the carotid body.

Carotid body expresses neutral endopeptidase (NEP)-like enzyme activity and phosphoramidon, an inhibitor of NEP augments sensory response of the carotid body to hypoxia (Kumar et al., 1990). NEP hydrolyzes substance P (SP) and methionine enkephalin (Met-ENK) in the nervous system. In the present study, we determined whether NEP hydrolyzes Met-ENK and SP in the carotid body and whether these peptides contribute to the phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anaesthetized adult cats. HPLC analysis showed that both SP and Met-ENK were hydrolyzed by the carotid body. Phosphoramidon (400 microM) markedly inhibited SP (approximately 90%) but had only marginal effect on Met-ENK hydrolysis (approximately 15%). Sensory responses of the carotid body in vitro to hypoxia (pO2, 68 +/- 6 mmHg) and SP (10 nmoles) were potentiated by phosphoramidon by approximately 80% and approximately 275% respectively (p < 0.01). SP-receptor antagonist abolished phosphoramidon-induced potentiation of the sensory response to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and SP plays a major role in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Animals↗

PubMed1999

NO and CO as second messengers in oxygen sensing in the carotid body.

It is being increasingly appreciated that nitric oxide (NO) and carbon monoxide (CO) are synthesized in mammalian cells and that they function as second messengers. The purpose of this article is to highlight the current information on NO and CO in the carotid body and discuss their significance in oxygen chemoreception. The NO synthesizing enzyme, nitric oxide synthase, is localized to nerve fibers and vascular endothelium in the carotid body. In vitro biochemical assays have shown that acute hypoxia inhibits NO synthase activity in carotid body extracts. Prolonged hypoxia up-regulates mRNA's encoding neuronal and endothelial NO synthases in the carotid body. Physiological studies have shown that NO is inhibitory to the carotid body sensory activity and mediates efferent inhibition. The actions of NO are in part mediated by its effects on glomus cells, wherein NO modulates Ca2+ channel activity and affects [Ca2+]i. The carotid body also uses another highly related gas as a second messenger, carbon monoxide (CO). The enzyme responsible for CO biosynthesis, heme oxygenase-2, is localized to glomus cells. CO, like NO, also exerts an inhibitory influence on sensory activity. Some of the actions of CO are mediated by altering Ca2+ channel activity and [Ca2+]i in glomus cells. Molecular oxygen is essential for biosynthesis of NO and CO. Under normoxia, basal levels of NO and CO act as amplifiers of molecular oxygen and keep the sensory discharge low. During hypoxia, decreased synthesis of NO and CO may contribute in part to the augmentation of sensory discharge.

Animals↗

PubMed1999

Norepinephrine inhibits a toxin resistant Ca2+ current in carotid body glomus cells: evidence for a direct G protein mechanism.

Previous studies have demonstrated that endogenous norepinephrine (NE) inhibits carotid body (CB) sensory discharge, and the cellular actions of NE have been associated with inhibition of Ca2+ current in glomus cells. The purpose of the present study was to elucidate the characteristics and mechanism of NE inhibition of whole cell Ca2+ current isolated from rabbit CB glomus cells and to determine the type(s) of Ca2+ channel involved. NE (10 microM) inhibited 24 +/- 2% (SE) of the macroscopic Ca2+ current measured at the end of a 25 ms pulse to 0 mV and slowed activation of the current. The alpha2 adrenergic receptor antagonist, SK&F 86466, attenuated these effects. Inhibition by NE was fast and voltage-dependent i.e., maximal at -10 mV and then diminished with stronger depolarizations. This is characteristic of G protein betagamma subunit interaction with the alpha1 subunit of certain Ca2+ channels, which can be relieved by depolarizing steps. A depolarizing step (30 ms to +80 mV) significantly increased (14 +/- 1%) current in the presence of NE, whereas it had no effect before application of NE (1 +/- 1%). To further test for the involvement of G proteins, NE was applied to cells where intracellular GTP was replaced by GDP-betaS. NE had little or no effect on Ca2+ current in cells dialyzed with GDP-betaS. To determine whether NE was inhibiting N- and/or P/Q-type channels, we applied NE in the presence of omega-conotoxin MVIIC (MVIIC). In the presence of 2.5 microM MVIIC, NE was equally potent at inhibiting the Ca2+ current (23 +/- 4% vs. 23 +/- 4% in control), suggesting that NE was not exclusively inhibiting N- or P/Q-type channels. NE was also equally potent (30 +/- 2% vs. 26 +/- 4% in control) at inhibiting the Ca2+ current in the presence of 2 microM nisoldipine, suggesting that NE was not inhibiting L-type channels. Further, NE inhibited a significantly larger proportion (47 +/- 6%) of the resistant Ca2+ current remaining in the presence of NISO and MVIIC. These results suggest that NE inhibition of Ca2+ current in rabbit CB glomus cells is mediated in most part by effects on the resistant, non L-, N-, or P/Q-type channel and involves a direct G protein betagamma interaction with this channel.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1999

Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism.

Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 microM, n = 7) and spermine nitric oxide (SNO; 100 microM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 microM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 microM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 microM LY 83, 583 (n = 7) nor was it mimicked by 600 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 microM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 microM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega-conotoxin GVIA, an N-type Ca2+ channel blocker (1 microM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO.

Aminoquinolines↗

PubMed1998

Role of c-fos in hypoxia-induced AP-1 cis-element activity and tyrosine hydroxylase gene expression.

Previous studies have demonstrated that hypoxia stimulates expression of the c-fos gene in intact animals and isolated cells. The purpose of the present study was to assess the functional significance of c-fos activation during hypoxia. Using antisense c-fos strategy, we tested the hypothesis that c-fos is essential for activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of down stream genes such as tyrosine hydroxylase (TH) gene during hypoxia. Experiments were performed on rat pheochromocytoma 12 (PC12) cells. AP-1 activity was determined by a reporter gene assay using a luciferase expression vector driven by two copies of an AP-1 cis-element (AP-1-Luc). Cells transfected with AP-1-Luc construct were exposed to normoxia (21% O2) or to varying intensities and/or durations of hypoxia. AP-1 activity increased in response to hypoxia. The magnitude of the response depended on the intensity and duration of the hypoxic stimulus. Increases in AP-1 activity could not be elicited in neuroblastoma cells, indicating that hypoxia-induced increase in AP-1 activity is a cell selective phenomenon. Antisense c-fos abolished hypoxia-induced AP-1 activation in PC12 cells. Hypoxia increased tyrosine hydroxylase-chloramphenicol acetyl transferase activity (TH-CAT), and antisense c-fos and mutations at AP-1 binding sites in TH promoter abolished this effect. These results provide direct evidence that c-fos is essential for functional activation of AP-1 and subsequent activation of delayed response genes such as TH in PC12 cells.

Animals↗

PubMed1998

Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase.

1. The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS-1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-1. 2. Experiments were performed on awake and anaesthetized mutant and wild-type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12% O2) and carbon dioxide (3 and 5% CO2 balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (VO2), CO2 production (VCO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3',5'-guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation. 3. Unanaesthetized mutant mice exhibited greater respiratory responses during 21 and 12% O2 than the wild-type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild-type mice. Changes in CO2 production and body temperature, however, were comparable between both groups of mice. 4. Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetized mutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12% O2, but not during 100% O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12% O2. 5. Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the response to hypoxia. 6. Respiratory responses to brief hyperoxia (Dejours test) and to cyanide, a potent chemoreceptor stimulant, were more pronounced in mutant mice, suggesting augmented peripheral chemoreceptor sensitivity. 7. cGMP levels were elevated in the brainstem during 21 and 12% O2 in wild-type but not in mutant mice, indicating decreased formation of nitric oxide in mutant mice. 8. The magnitude of respiratory responses to hypercapnia (3 and 5% CO2 balanced oxygen) was comparable in both groups of mice in the awake and anaesthetized conditions. 9. These observations suggest that the hypoxic responses were selectively augmented in mutant mice deficient in NOS-1. Peripheral as well as central mechanisms contributed to the altered responses to hypoxia. These results support the idea that nitric oxide generated by NOS-1 is an important physiological modulator of respiration during hypoxia.

Animals↗

PubMed1998

Carotid body I1-imidazoline receptors: binding, visualization and modulatory function.

The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I1-imidazoline and alpha2-adrenergic receptors, which also recognize imidazolines, specific [125I]p-iodoclonidine binding to carotid body membranes was characterized. The specific alpha2-agents epinephrine (100 microM) or SK&F 86466 (10 microM) inhibited only a portion of specific [125I]p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I1-imidazoline as well as alpha2-adrenergic sites. The distribution of [125I]p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [125I]p-iodoclonidine, with both I1-imidazoline and alpha2-adrenergic sites expressed. The relevance of I1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after alpha2-adrenergic blockade. Clonidine (100 microg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of alpha2-adrenergic receptors.

Adrenergic alpha-Agonists↗

PubMed1998

Endogenous carbon monoxide in control of respiration.

It is being increasingly appreciated that gas molecules such as nitric oxide (NO) function as chemical messengers in the nervous system. Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as NO. The purpose of this article is to highlight the current information on the significance of endogenously generated CO in control of breathing. In mammalian cells, CO is generated during oxidative cleavage of heme by heme oxygenases (HO) and molecular oxygen is essential for this reaction. Two forms of HO have been identified including an inducible HO-1, that resembles stress-inducible protein HSP-32, and a constitutively expressed HO-2. HO-2 is expressed in many respiratory related neural structures including airway ganglion, carotid body, petrosal and nodose ganglia., nucleus of the tractus solitarius (nTS), and neurons of the rostral ventrolateral medulla (RVLM). Basal expression of HO-1 is either very low or even absent, but can be elevated during oxidative stress and hypoxia. Physiological studies have shown that CO might be of importance in vagally mediated contractions of airways. Several lines of evidence indicate that endogenously generated CO is a physiological modulator of the ventilatory response to hypoxia via its actions on carotid bodies and perhaps at brainstem neurons. In addition, CO might play a role in ventilatory adaptation to hypoxia, as low oxygen is a potent inducer of HO-1. Many of the neuronal structures that express HO also contain NOS, the enzyme that generates NO. Much remains to be studied on regulatory interactions between CO and NO and their impact on breathing.

Brain↗

PubMed1998

Release of dopamine and norepinephrine by hypoxia from PC-12 cells.

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (PO2 of medium approximately 40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of PO2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was approximately 11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 microM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in PO2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

Animals↗

PubMed1997

Ca2+ current in rabbit carotid body glomus cells is conducted by multiple types of high-voltage-activated Ca2+ channels.

Ca2+ current in rabbit carotid body glomus cells is conducted by multiple types of high-voltage-activated Ca2+ channels. J. Neurophysiol. 78: 2467-2474, 1997. Carotid bodies are sensory organs that detect changes in arterial oxygen. Glomus cells are presumed to be the initial sites for sensory transduction, and Ca2+-dependent neurotransmitter release from glomus cells is believed to be an obligatory step in this response. Some information exists on the Ca2+ channels in rat glomus cells. However, relatively little is known about the types of Ca2+ channels present in rabbit glomus cells, the species in which most of the neurotransmitter release studies have been performed. Therefore we tested the effect of specific Ca2+ channel blockers on current recorded from freshly dissociated, adult rabbit carotid body glomus cells using the whole cell configuration of the patch-clamp technique. Macroscopic Ba2+ current elicited from a holding potential of -80 mV activated at a Vm of approximately -30 mV, peaked between 0 and +10 mV and did not inactivate during 25-ms steps to positive test potentials. Prolonged ( approximately 2 min) depolarized holding potentials inactivated the current with a V1/2 of -47 mV. There was no evidence for T-type channels. On steps to 0 mV, 6 mM Co2+ decreased peak inward current by 97 +/- 1% (mean +/- SE). Nisoldipine (2 mu M), 1 mu M omega-conotoxin GVIA, and 100 nM omega-agatoxin IVa each blocked a portion of the macroscopic Ca2+ current (30 +/- 5, 33 +/- 5, and 19 +/- 3% after rundown correction, respectively). Simultaneous application of these blockers revealed a resistant current that was not affected by 1 mu M omega-conotoxin MVIIC. This resistant current constituted 27 +/- 5% of the total macroscopic Ca2+ current. Each blocker had an effect in every cell so tested. However, the relative proportion of current blocked varied from cell to cell. These results suggest that L, N, P, and resistant channel types each conduct a significant proportion of the macroscopic Ca2+ current in rabbit glomus cells. Hypoxia-induced neurotransmitter release from glomus cells may involve one or more of these channels.

Animals↗

PubMed1996

Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons.

In the present study we examined the effects of hypobaric hypoxia on neuronal (n) and endothelial (e) nitric oxide synthase (NOS) gene expression in the central and peripheral nervous system. Adult rats were exposed either to normoxia (room air) on to hypobaric hypoxia (0.4 atm) for 4, 12 or 24 h and cerebellum and nodose ganglion representing the central and peripheral neurons, respectively, were removed. Messenger RNAs encoding n- and eNOS as well as beta-actin were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) technique. Hypoxia increased nNOS mRNA expression with maximal changes occurring after 12 h wherein mRNA levels were increased by 10.4 +/- 1.3 and 2 +/- 0.4 fold in nodose ganglion and cerebellum, respectively. Hypoxia, on the other hand, had no significant effect on eNOS and beta-actin mRNA levels. Analysis of nNOS protein and enzyme activity showed near doubling of these variables in both tissues after 24 h of hypoxia, indicating that nNOS protein levels are increased and that the protein is functionally active. These observations demonstrate that 12-24 h of hypobaric hypoxia selectively activates nNOS gene expression, which is reflected in an increase in nNOS protein in central and peripheral neurons. It is suggested that up-regulation of nNOS leads to increased generation of nitric oxide, which in turn may contribute to the readjustments of cardio-respiratory systems during the early stages of chronic hypoxia.

Animals↗