β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

Stoichiometry of the rat kidney Na+-HCO3 – cotransporter expressed in Xenopus laevis oocytes

The rat kidney Na⁺-HCO₃ ^– cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na⁺ and HCO₃ ^– as permeable ions. The current carried by rkNBC (I _NBC) was identified by its response to changing bath Na⁺ concentration(s) and quantified as the current blocked by 4,4’-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO₃ ^– to Na⁺ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 μmol/l) blocks I _NBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na⁺ and HCO₃ ^– concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO₃ ^– concentration was 25 mmol/l throughout, but the Na⁺ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch. Received: 19 February 1999 / Received after revision: 5 March 1999 / Accepted: 6 March 1999     

Postnatal expression of α2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of α and β subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of α3, α5, α7, and β4 subunit mRNAs and α7 binding sites during hippocampal and cortical development. Here, we examined the expression of α2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. α2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of α2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of α2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, α2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, α2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing α2 subunits could regulate GABAergic activity during a critical period of network formation.     

β-Subunits co-determine the sensitivity of rat neuronal nicotinic receptors to antagonists

We have investigated the effect of 4 ganglionic cholinergic antagonists (hexamethonium, mecamylamine, pentolinium, trimetaphan) on rat 32 and 34 neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. Current responses were elicited by fast application of acetylcholine on voltage-clamped oocytes (holding potential Vinh = -80mV). Concentration-inhibition curves were used to get estimates of IC₅₀, the antagonist concentration yielding 50% reduction of the peak current. The K_B's of the antagonists were calculated using estimates of the apparent K_D of acetylcholine. The order of affinity of the antagonists was similar for both receptor subtypes: mecamylamine pentolinium > hexamethonium > trimetaphan. However, 34 neuronal nAChRs were 9 to 22 times more sensitive to each of the 4 antagonists than 32 receptors. These results further underline the importance of the -subunit as co-determinant of the functional properties of neuronal nAChRs.     

Potential roles of recombinant acetylcholine receptor α subunit 1-211 in immunoadsorbent and DNA immunization

The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChR(α211) immunization.     

Spatial and intracellular relationships between the α7 nicotinic acetylcholine receptor and the vesicular acetylcholine transporter in the prefrontal cortex of rat and mouse

The alpha 7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the α7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the α7nAChR and VAChT. In both species, the α7nAChR-immunoreactivity (⁻ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable α7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of α7nAChR-labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n=240). Of the α7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic α7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed α7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the α7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.     

Combined blockade of α3β4 nicotinic acetylcholine receptors and GluR1 AMPA receptors in rats prevents kainate-induced tonic-clonic seizures

Intramuscular injection of IEM-1754, a blocker of cerebral GluR1 AMPA receptors, in doses of 0.5–3.0 mg/kg decreased the incidence of kainate-induced tonic-clonic seizures and mortality rate by 2.7–4 times. IEM-1678, an α3β4 nicotinic acetylcholine receptor antagonist administered intramuscularly in a maximum dose of 3 mg/kg decreased the incidence of kainate-induced tonic-clonic seizures and mortality rate by 2.3–2.7. IEM-1460 blocking both GluR1 AMPA receptors and α3β4 nicotinic acetylcholine receptors, injected intramuscularly in doses of 0.5–3.0 mg/kg produced the maximum anticonvulsant activity and 8-fold decreased the incidence of kainate-induced tonic-clonic seizures and mortality rate. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 5, pp. 548–550, May, 2007     

Expression of estrogen receptors α and β in the trigeminal mesencephalic nucleus of adult women and men

Temporomandibular disorders are more prevalent in women than in men and phases of pain relate to the estrous cycle. Several studies described the location of estrogen receptors (ER) in the temporomandibular joint (TMJ), the masseteric muscles and cartilage, but it was unknown whether they are also expressed within the pseudounipolar neurons of the trigeminal mesencephalic nucleus, which receives direct sensory inputs from these structures. Therefore, we studied expression of ERα and ERβ protein in the trigeminal mesencephalic nucleus of ten human brains (five female/five male). Both receptors were uniformly expressed on neurons, but not other cell types within the target structure. Thus, sensory inputs from the TMJ and adjacent structures are likely to be modulated by estrogen at the level of the first sensory neuron which may underlie the well-known correlation of pain incidence and phases of the estrous cycle.     

The role of the acetylcholine — Cholinesterase system in the origin of bioelectrical phenomena in skeletal muscle

Summary Poisoning of the isolated sartorius muscle in frog with eserine and strychnine in the 110⁵ and 110⁴ concentrations causes the change of the value and the form of the excitation biocurrent. Strychnine and eserine in the 110⁵, 110⁴ and 110³ concentrations have no effect on the electromotive force of the sartorius muscle injury. These data lead us to presume certain differences in the mechanism of the origin of the excitation and injury biocurrents in the skeletal muscle, at least with relation to the participation of the acetylcholine-cho-linesterase system in this process.Presented by Academician the late L. A. Orbeli     

PIKing the right isoform: the emergent role of the p110β subunit in breast cancer

Class IA phosphoinositide-3’-kinases (PI3Ks) regulate many cellular processes. Despite a clear implication of PI3K in cancer, the involvement of each of its isoforms namely p110α and p110β in the development of breast cancer remains elusive. Until recently, the spotlight was given to the α subunit; however, the p110β isoform has now emerged as an interesting target as well. In order to determine the importance of both these subunits in breast cancer, we aimed to study the expression of p110α and p110β in a series of invasive breast carcinomas. We constructed tissue microarrays from 315 invasive breast carcinomas and performed immunohistochemistry for p110α and β, correlating the expression patterns with clinicopathological parameters. Furthermore, overall survival was analysed through Kaplan–Meier survival curves and Cox regression. We found that p110 subunits are expressed in 23.8% of invasive breast carcinomas, of which 11.8% express p110α and 15.2% p110β. The p110α positive tumours correlated with hormone receptor (HR) expression, and were not associated with overall survival. The membrane expression of p110β was associated with worse prognosis. This was due to its link to HER2-overexpression, lower age of onset, higher grade, lymph node involvement, distant metastasis and was inversely associated with HR status. Furthermore, p110β expression was associated with worse overall survival. Importantly our results indicate a role for the beta subunit in the development/progression of HER2-overexpressing tumours, highlighting possible therapeutic associations between HER2 and p110β inhibitors.     

What is the role of Toll-like receptors in bacterial infections?

Innate immunity relies on signalling by Toll-like receptors (TLRs) to alert the immune system of the presence of invading bacteria. TLR activation leads to the release of cytokines that allow for effective innate and adaptive immune responses. However, the contribution of different TLRs depends on the site of the infection and the pathogen. This review will describe the involvement of TLRs in the development of three different bacterial infections as well as our current understanding of the role of TLRs during microbial pathogenesis.     

Neuronal basis for the hyperthermic effect of μ-opioid agonists in rats: decrease in temperature sensitivity of warm-sensitive hypothalamic neurons

The effect of the selective μ-opioid receptor agonist Tyr-Pro-N-MePhe-d-Pro-NH₂ (PL-017) on tonic activity and temperature sensitivity of neurons in the preoptic area/anterior hypothalamus (PO/AH) has been examined in rat brain slices using extracellular recordings. The tonic activity of both warm-sensitive and temperature-insensitive neurons was inhibited in a dose-dependent manner by superfusion with the μ-agonist (0.5–100 nM). The temperature sensitivity was selectively decreased in warm-sensitive neurons in concentrations up to 10 nM. Only in the high concentration of 100 nM did the μ-agonist reduce the temperature coefficient of both types of neurons. Pretreatment with equimolar concentrations of the μ-antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP) prevented the effects of the μ-agonist (0.5–10 nM) on tonic activity as well as temperature sensitivity. The results suggest that a specific change of the temperature sensitivity of PO/AH neurons is involved in the hyperthermia induced by μ-agonists in rats.     

Targeting α-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury

We have previously shown that vagal nerve stimulation prevents intestinal barrier loss in a model of severe burn injury in which injury was associated with decreased expression and altered localization of intestinal tight junction proteins. α-7 Nicotinic acetylcholine receptor (α-7 nAchR) has been shown to be necessary for the vagus nerve to modulate the systemic inflammatory response, but the role of α-7 nAchR in mediating gut protection remained unknown. We hypothesized that α-7 nAchR would be present in the gastrointestinal tract and that treatment with a pharmacological agonist of α-7 nAchR would protect against burn-induced gut barrier injury. The effects of a pharmacological cholinergic agonist on gut barrier integrity were studied using an intraperitoneal injection of nicotine 30 minutes after injury. Intestinal barrier integrity was examined by measuring permeability to 4-kDa fluorescein isothiocyanate-dextran and by examining changes in expression and localization of the intestinal tight junction proteins occludin and ZO-1. Nicotine injection after injury prevented burn-induced intestinal permeability and limited histological gut injury. Treatment with nicotine prevented decreased expression and altered localization of occludin and ZO-1, as seen in animals undergoing burn alone. Defining the interactions among the vagus nerve, the enteric nervous system, and the intestinal epithelium may lead to development of targeted therapeutics aimed at reducing gut barrier failure and intestinal inflammation after severe injury.     

Estrogens regulate neuroinflammatory genes via estrogen receptors α and β in the frontal cortex of middle-aged female rats

Background

Increasing evidence links diverse forms of air pollution to neuroinflammation and neuropathology in both human and animal models, but the effects of long-term exposures are poorly understood.

Objective

We explored the central nervous system consequences of subchronic exposure to diesel exhaust (DE) and addressed the minimum levels necessary to elicit neuroinflammation and markers of early neuropathology.

Methods

Male Fischer 344 rats were exposed to DE (992, 311, 100, 35 and 0 μg PM/m³) by inhalation over 6 months.

Results

DE exposure resulted in elevated levels of TNFα at high concentrations in all regions tested, with the exception of the cerebellum. The midbrain region was the most sensitive, where exposures as low as 100 μg PM/m³ significantly increased brain TNFα levels. However, this sensitivity to DE was not conferred to all markers of neuroinflammation, as the midbrain showed no increase in IL-6 expression at any concentration tested, an increase in IL-1β at only high concentrations, and a decrease in MIP-1α expression, supporting that compensatory mechanisms may occur with subchronic exposure. Aβ42 levels were the highest in the frontal lobe of mice exposed to 992 μg PM/m³ and tau [pS199] levels were elevated at the higher DE concentrations (992 and 311 μg PM/m³) in both the temporal lobe and frontal lobe, indicating that proteins linked to preclinical Alzheimer's disease were affected. α Synuclein levels were elevated in the midbrain in response to the 992 μg PM/m³ exposure, supporting that air pollution may be associated with early Parkinson's disease-like pathology.

Conclusions

Together, the data support that the midbrain may be more sensitive to the neuroinflammatory effects of subchronic air pollution exposure. However, the DE-induced elevation of proteins associated with neurodegenerative diseases was limited to only the higher exposures, suggesting that air pollution-induced neuroinflammation may precede preclinical markers of neurodegenerative disease in the midbrain.     

Effect of nicotine on mRNA levels encoding opioid peptides,vasopressin and α3 nicotinic receptor subunit in the rat

Summary The effect of acute and chronic nicotine treatment of rats on the mRNA levels coding for the three opioid peptide precursors, for provasopressin and for the a3 subunit of nicotinic receptors in brain, pituitary and/or adrenal medulla of rats was investigated. Nicotine was found to increase the levels of proenkephalin mRNA in the adrenal medulla, but did not affect the levels of PENK mRNA in striatum, hypothalamus and hippocampus. The mRNA levels of prodynorphin were increased together with that of provasopressin in the hypothalamus after nicotine, whereas the prodynorphin mRNA levels in the hippocampus and the striatum remained unchanged. Nicotine treatment resulted in an increase in the pro-opiomelanocortin mRNA levels in the anterior pituitary and in a decrease in the intermediate pituitary, but did not change the levels of pro-opiomelanocortin mRNA in the hypothalamus. The levels of mRNA coding for the ₃ subunit of nicotinic receptors in the hypothalamus and the adrenal medulla remained unchanged. The increase in the prodynorphin and provasopressin mRNA levels in the hypothalamus was most pronounced 1 day after s.c. application of two doses of 0.4 mg/kg nicotine (about 100% above control). A smaller increase in mRNA concentrations (about 30%) was found after tonic infusion of the drug for 4 days (4 mg/kg per day), whereas no change was observed after tonic infusion of nicotine for 7 and 14 days indicating the development of complete tolerance. The increase in proenkephalin mRNA levels in the adrenal medulla was highest after the short-term application of nicotine (about 150% above control). Less, but still significant increases in the mRNA levels (about 40%) were also seen after 7 and 14 days of tonic nicotine administration suggesting that no complete desensitization is developing to the effect of nicotine. This desensitization appeared to be less pronounced when the drug was applied in a pulsatile manner using minipumps, since a high increase in the PENK mRNA levels (100% above control levels) was observed after intermittent infusion of nicotine (six boli per day of 1 mg/kg for 7 days). These findings demonstrate that nicotine can alter gene expression of opioid peptides and vasopressin in certain rat tissues, and that the mode of drug administration plays an important role in this effect of nicotine. The possibility that the pulsatile administration of nicotine by cigarette smokers may also result in an altered expression of opioid peptide genes in humans is discussed.Abbreviations PENK proenkephalin - POMC pro-opiomelanocortin - MSH melanocyte stimulating hormone - PDYN prodynorphin - CRF corticotropin releasing factor     

The role of T-cell receptor α and β genes in MHC-restricted antigen recognition

T cells use α- and β-chain genes, which are organized and diversified by somatic DNA rearrangements much like the immunoglobulin genes, to encode clonally distributed receptor molecules which confer specificity for MHC and antigen. Here Zlatko Dembic and his colleagues summarize recent mutagenesis and gene transfection experiments indicating that α- and β-chain genes are necessary and sufficient for T-cell specificity.