Ozra Dehkordi

Neuronal expression of bitter taste receptors and downstream signaling molecules in the rat brainstem

Previous studies have shown that molecules of the taste transduction pathway may serve as biochemical markers for chemoreceptive cells in respiratory and gastrointestinal tracts. In this study, we tested the hypothesis that brainstem neurons contain signaling molecules similar to those in taste buds which may sense the chemical composition of brain extracellular fluids. We used the reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemical techniques to evaluate presence of different bitter-responsive type 2 taste receptors (T2Rs), their associated G-protein α-gustducin, the downstream signaling molecules phospholipase C isoform β2 (PLC-β2) and transient receptor potential melastatin 5 (TRPM5) in the brainstem of rats. RT-PCR confirmed the mRNA coding for α-gustducin, PLC-β2, TRPM5 and rT2R1 but not that of rT2R16, rT2R26 and rT2R38 in the medulla oblongata. Western blotting confirmed the presence of α-gustducin at the protein level in rat brainstem. Immunohistochemistry identified cells expressing α-gustducin and PLC-β2 at multiple cardiorespiratory and CO(2)/H(+) chemosensory sites, including rostral ventral medulla, facial, parapyramidal, solitary tract, hypoglossal and raphe nuclei. In the medullary raphe, α-gustducin and PLC-β2 were colocalized with a subpopulation of tryptophan hydroxylase (TPH)-immunoreactive serotonergic neurons, a subset of which has respiratory CO(2)/H(+) chemosensitivity. Presence of the T2R1 gene and other genes and proteins of the bitter taste transduction pathway in the brainstem implies additional functions for taste receptors and their effector molecules apart from their gustatory function.

Expression of α-7 nAChRs on spinal cord-brainstem neurons controlling inspiratory drive to the diaphragm

In the present study, we determined whether alpha-7 subunit containing nicotinic acetylcholine receptors (nAChRs) are expressed by neurons within the pre-Botzinger complex (pre-BotC), bulbospinal, and phrenic motor nuclei in the rat. alpha-7 Immunohistochemistry combined with cholera toxin B (CTB), a retrograde tracer was used to detect expression of alpha-7 nAChRs by phrenic motor and bulbospinal neurons. Neurokinin-1 receptor immunoreactivity was used as a marker for pre-BotC neurons. Of the CTB-positive neurons in the phrenic nuclei, 60% exhibited immunoreactivity for alpha-7 nAChRs. Of the bulbospinal neurons in the paramedian reticular nuclei (PMn), gigantocellular nuclei (Gi), raphe nuclei, rostral ventrolateral medulla (RVLM) and nucleus tractus solitarius, 20-50% were found to express alpha-7 nAChR immunoreactivity. Of the peudorabies virus (PRV) labeled bulbospinal neurons in PMn, Gi, raphe and RVLM, 9-12% co-expressed alpha-7 nAChRs. Immunoreactivity for alpha-7 nAChRs was also detected in 57% of the neurokinin-1 receptor containing neurons presumed to reside in pre-BotC. These findings suggest that nicotinic cholinergic regulation of the chest wall pumping muscles may occur at multiple levels of the central nervous system.

Co-expression of nAChRs and molecules of the bitter taste transduction pathway by epithelial cells of intrapulmonary airways.

AIMS The ability to sense the bitter taste of nicotine is an important component of addiction to, and withdrawal from, cigarette smoking. alpha-Gustducin and phospholipase C-beta2 (PLC-beta2), molecules involved in the taste transduction pathway, have been identified in airway epithelial solitary chemosensory cells (SCCs). Airway epithelial cells also express multiple nicotinic acetylcholine receptors (nAChRs). However, the relationship between nAChRs and molecules of taste transduction in response to nicotine is not known. This study was designed to determine whether nAChRs and the taste transduction molecules alpha-gustducin, PLC-beta2 and bitter taste receptors (T2R38) reside at sites of the intrapulmonary airways where interaction with the nicotine components of cigarette smoke is likely. MAIN METHODS We used the reverse transcription-polymerase chain reaction (RT-PCR) to detect alpha-gustducin, PLC-beta2 and T2R38 mRNA and immunohistochemistry to localize expression of these proteins by nAChR expressing cells of the airway. KEY FINDINGS RT-PCR demonstrated the presence of mRNA for alpha-gustducin, PLC-beta2 and T2R38. Immunohistochemistry showed the expression of alpha-gustducin, PLC-beta2 and T2R38 by subsets of epithelial cells at all levels of the intrapulmonary airways including bronchi, terminal and respiratory bronchioles. Double labeling demonstrated the co-expression of alpha-gustducin with alpha3, alpha4, alpha5, alpha7 and beta2, as well as, PLC-beta2 and T2R38 with alpha4, alpha5 and beta2 nAChR subunits. SIGNIFICANCE These findings provide morphological evidence for the presence of molecules of the bitter taste transduction pathway in nAChR expressing SCCs of the intrapulmonary airways. These SCCs may, thus, constitute a peripheral component of the bitter taste signal transduction pathway for nicotine.

Expression of alpha-7 and alpha-4 nicotinic acetylcholine receptors by GABAergic neurons of rostral ventral medulla and caudal pons

Rostral ventral medulla (RVM) contains significant numbers of local GABAergic neurons which may subserve respiratory chemosensory and baroreceptor reflexes. Nicotinic mechanisms stimulate release of GABA in certain brainstem neurons. Whether the GABAergic neurons at RVM express nicotinic cholinergic receptors (nAChRs) is not known. We used glutamic acid decarboxylase 67-kDa isoform (GAD67) and parvalbumin (PV) as anatomical markers to identify the GABAergic neurons of the RVM and caudal pons and performed double labeling to evaluate the expression of alpha-7 and alpha-4 nAChRs by GAD67 and PV-imnunoreactive (ir) cells at these sites. GAD67-ir cells were found at the ventrolateral pontomedullary border in areas adjacent to the A5 noradrenergic cell group and overlapping the facial nucleus lateral subnuclei and para-facial zones. Of 205 GAD67-ir cells labeled at these sites, 74% exhibited immunoreactivity for alpha-7 nAChRs. Alpha-4 immunoreactivity was also present in 35% of GAD67-ir cells at these sites. The PV-ir cells of RVM and caudal pons were found medial to the facial nucleus and lateral to the pyramid in a column distinct from the GAD67-ir cells. Virtually all the PV-ir cells demonstrated immunoreactivity for alpha-4 nAChR (95%) and alpha-7 (93%) subunits of nAChRs. Differential expression of GAD67 and PV by neurons at the pontomedullary border implies that PV may not be a valid marker for GABAergic neurons. The expression of alpha-4 and alpha-7 nAChRs by GAD67-ir cells suggests nicotinic cholinergic modulation of GABAergic signaling at these ventrolateral pontomedullary sites.

Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons

In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency.

Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.

Analgesic responses to intrathecal morphine in relation to CSF concentrations of morphine-3,β-glucuronide and morphine-6,β-glucuronide

Abstract This study was performed to determine whether variations in analgesic responses to intrathecal morphine could be explained by cerebrospinal fluid (CSF) concentrations of morphine metabolites. Twenty-four CSF samples were collected at the beginning, middle and end of treatment periods in seven cancer patients with pain of malignant origin. CSF concentrations of morphine-3,β-glucuronide (M3G) and morphine-6,β-glucuronide (M6G) metabolites were measured by gas chromatography/mass spectrometry. Analgesic responses to morphine were estimated concurrent with CSF collection using a visual analog scale representing percentages of pain relief. Effective analgesia was defined as ≥ 75% pain relief. CSF concentration of M3G and M6G in the 24 samples were 722 ± 116 ng/ml and 699 ± 158 ng/ml, respectively. CSF samples were categorized into two groups: (1) those collected during effective analgesia (N = 14), and (2) those collected during ineffective analgesia (N = 10). M6G levels detected in group 1 samples (effective analgesia) were significantly greater than those found in group 2 samples (ineffective analgesia) (978 ± 243 ng/ml vs 309 ± 68 ng/ml, P M3G M6G ratios were not significant. It is concluded that CSF M6G may be indicative of effectiveness of analgesia in cancer patients subjected to intrathecal morphine.

Somatosensory evoked potential, neurological examination and magnetic resonance imaging for assessment of cervical spinal cord decompression.

The present study was designed to determine the relationship between neurological testing, anatomical imaging, and electrophysiological monitoring for assessing outcome of cervical spinal cord decompression. We prospectively studied 28 consecutive patients (age 39-76 yr) who were subjected to presurgical-(1-3 wk) and postsurgical (3-4 mo) neurological examination and recording of the median nerve somatosensory evoked potential (SEP). In 13 patients, magnetic resonance imaging (MRI) was also performed. Changes in neurological function, SEP and MRI were evaluated and graded as (1) improvement,(2) no change or (3) deterioration. Neurological outcome (NO) was based on changes in motor grade strength, sensory, reflexes and gait. The SEP outcome was based on changes in latency and disappearance of SEP waveform components whereas MRI evaluation was based on changes in spinal cord and canal diameters. Significance of association between NO, SEP and MRI was determined by Pearsons Chi-Square statistic (P<.05). The SEP improved in 71% (20/28) and deteriorated in 28% (8/28) of the subjects. An association between SEP changes and NO was found in 82% (23/28) of the subjects (P = .0038). Decompression increased the spinal canal diameter in 92% (12/13), and the spinal cord diameter in 38% (5/13) of the subjects. An association between NO, or SEP and MRI was not detected. Changes in median nerve SEP latency appear to be predictive of the neurological status of patients subjected to cervical spinal cord decompression. Postoperative increments in SEP latency or disappearance of the SEP waves were indicative of poor outcome after surgical decompression of the cervical spinal cord.

Neuroanatomical Relationships of Substance P-Immunoreactive Intrapulmonary C-Fibers and Nicotinic Cholinergic Receptors

Previous studies have suggested that sensory mechanisms may be important components of addiction to, and withdrawal from, cigarette smoking. The sensory and respiratory responses to nicotine are mediated, in part, by bronchopulmonary C‐fiber afferents. Nicotine has a direct stimulatory effect on pulmonary sensory neurons, and nicotinic cholinergic receptors (nAChRs) composed of various combinations of α and β subunits are known to be present in pulmonary ganglia. At the subcellular level, however, little is known about expression of nAChRs on sensory fibers in the intrapulmonary airways. The present study was therefore designed to evaluate the expression of nAChRs on a subset of intrapulmonary sensory nerve endings known to exhibit immunoreactivity for substance P (SP). The presence of nAChR subunits was first confirmed at the mRNA and protein levels in rat lung tissues by using RT‐PCR and Western blot techniques. Then, double labeling of SP‐immunoreactive (‐IR) C‐fibers and different nAChR subunits was performed. α2, α3, α4, α5, α7, and β2 subunits were detected at all levels of the intrapulmonary airways; including bronchi, terminal and respiratory bronchioles, alveolar walls, and alveolar macrophages. None of the nAChR subunits studied was expressed by the SP‐IR C‐fibers. However, SP‐expressing C‐fibers were observed in close proximity to and intermingling with nAChR‐expressing airway epithelial cells. The close proximity of C‐fibers to nAChR‐expressing airway epithelial cells suggests that a component of nicotinic stimulation of SP‐IR C‐fiber afferents may be mediated by endogenous chemical substances released by nAChR‐expressing epithelial cells.

Growth inhibition of subcutaneously transplanted hepatomas without cachexia by alteration of the dietary arginine‐methionine balance

Previous studies have shown that alteration of the dietary arginine-methionine balance by use of synthetic L-amino acids inhibits tumor growth of a subcutaneously transplanted Morris hepatoma at the expense of maintaining body weight. However, L-methionine is susceptible to degradation and, therefore, may contribute to a deficiency state. The present studies were performed to determine whether growth of subcutaneous hepatoma transplants is inhibited, and body growth maintained, when rats are fed diets containing L-methionine in replacement of N-acetyl-L-methionine (NALM) for 28 days. Tumor-free and tumor-bearing rats fed a control diet, with amino acids replacing protein, had gains in body weight: 31.3 +/- 1.0 and 19.1 +/- 0.5 g (12% and 7%), respectively. Rats fed six experimental diets, with varying L-arginine-NALM balances, had body weight gains ranging from 18.4 +/- 0.3 to 26.7 +/- 0.9 g (7-10%). Tumor weight of control rats was 10.65 +/- 0.24% of body weight. Diets supplemented with L-arginine in combination with normal and deficient NALM decreased tumor weights by 35% and 38%, respectively, It is concluded that dietary replacement of L-methionine with NALM and supplementation with L-arginine inhibits growth of a subcutaneously transplanted Morris hepatoma in the absence of cachexia.