Specific contribution of human T-type calcium channel isotypes (α1G, α1H and α1I) to neuronal excitability

The glutamine transporter SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of system N amino acid transport. Two different transport mechanisms have been proposed for this transporter. These are an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na⁺ and 1H⁺, or an electrogenic mechanism coupled to the exchange of 2Na⁺ against 1H⁺. This study was performed to solve these discrepancies and to investigate the reversibility of the transporter. When SN1 was expressed in Xenopus laevis oocytes, glutamine uptake was accompanied by a cotransport of 2–3 Na⁺ ions as determined by ²²Na⁺ fluxes. However, at the same time a rapid release of intracellular Na⁺ was observed indicating an active exchange of Na⁺ ions. The driving force of the proton electrochemical gradient was equivalent to that of the sodium electrochemical gradient. Acidification of the extracellular medium caused the transporter to run in reverse and to release glutamine. Determination of accumulation ratios at different driving forces were in agreement with an electroneutral 1Na⁺-glutamine cotransport-1H⁺ antiport. Inward currents that were observed during glutamine uptake were much smaller than expected for a stoichiometric cotransport of charges. A slippage mode in the transporter mechanism and pH-regulated endogenous oocyte cation channels are likely to contribute to the observed currents.     

The involvement of Cav3.2/α1H T-type calcium channels in excitability of mouse embryonic primary vestibular neurones

Ca²⁺ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Ca_v3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between −65 and −45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Ca_v3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni²⁺ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Ca_v3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Ca_v3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Immunostaining for α1-antichymotrypsin and α1-antitrypsin in gliomas

Antisera to α₁-antichymotrypsin, α₁-antitrypsin and lysozyme were reacted with 20 cases of glioblastoma multiforme, seven anaplastic astrocytomas, eight astrocytomas, six oligodendrogliomas, four ependymomas and the cerebral cortex from six normal autopsy brains. In addition, two pleomorphic xantho-astrocytomas and two heavily lipidized malignant gliomas were similarly examined. All astrocytic lesions were confirmed with anti-GFAP antisera. Thirty astrocytic tumours (77%), four oligodendrogliomas (67%) and three ependymomas (75%) reacted positively with anti-α₁-antichymotrypsin; 25 astrocytic tumours (64%), three oligodendrogliomas (50%) and three ependymomas (75%) showed positive staining for α₁-antitrypsin. The pattern of staining with either of these two markers did not correlate with tumour grading. None of the gliomas examined stained positively with anti-lysozyme. Non-neoplastic glial elements did not react with any of the three antisera. The results of this study suggest that staining for α₁-antichymotrypsin and α₁-antitrypsin is of little value in the differential diagnosis of neuroepithelial or mesenchymal lesions in the brain.     

Gastric motility in soluble guanylate cyclase α1 knock-out mice

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α₁β₁-isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα₁ knock-out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non-nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real-time RT-PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild-type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1 H [1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxations evoked by exogenous NO and the NO-independent sGC activator BAY 41-2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8-bromo-cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41-2272 still induced a moderate ODQ-sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα₁β₁ plays an important role in gastric nitrergic relaxation in vitro , but some degree of nitrergic relaxation can occur via sGCα₂β₁ activation in sGCα₁ KO mice, which contributes to the moderate in vivo consequence on gastric emptying.     

13.α 1-Fetoprotein

Summary. Rocket immunoelectrophoresis suitable for both quantitation and qualitative identification of α₁-fetoprotein is presented. By a slight modification of the rocket immunoelectrophoresis it is possible to detect α₁-fetoprotein in serum in a concentration of about 0.1 mg/1. A simple method for the final purification of α₁-fetoprotein is described. The concentration of α₁-fetoprotein in cord blood from 258 newborn infants is shown to be closely related to the gestational age of the newborn infants. The confidence limits for the estimation of gestational age are± 16 days.     

Heterozygous α-antichymotrypsin and PiZ (α1-antitrypsin deficiency

In a case-control study we compared the prevalence of heterozygous deficiency of two closely related anti-neutrophil protease inhibitors, alpha 1-antitrypsin and alpha 1-antichymotrypsin, in 172 consecutive children with asthma. In a cohort study the clinical spectrum and severity were compared. On the basis of family studies 5/172 (2.9%) were classified as heterozygotes for alpha 1-antichymotrypsin deficiency, a high prevalence compared with that of an unselected adult population (prevalence ratio 4.5 (1.7-11.9), P less than 0.005). This finding suggests that the carrier state of this rare allele (prevalence 0.64%) may predispose to asthma in children. Among these heterozygous patients the prevalence of positive RAST tests for foodstuffs was significantly increased (prevalence ratio 4.8 (1.7-13.2), P less than 0.005) and 2/5 manifested food allergy with Quincke oedema. Either the PiMZ or SZ phenotype of alpha 1-antitrypsin deficiency was found in 12 (7.0%) of the 172 patients, a prevalence similar to that of a normal population (prevalence ratio 1.3 (0.67-2.6), P = 0.44). However, the asthma was more severe among the Z allele carriers, judged by the number of hospital admissions, compared with the non-Z asthmatic children (mean 2.92 vs. 1.72, P less than 0.05). The results indicate that heterozygous deficiency of protease inhibitors directed against neutrophil proteases may affect the severity and clinical spectrum of childhood asthma, and to some degree be predisposing.     

Immunosuppressive Properties of α1-Microglobulin

α₁-Microglobulin (α₁m), a serum glycoprotein (26,000 d). was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that, α₁m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of α₁m on antigen stimulation did not seem to be due to binding of α₁m to PPD or cells, to altered kinetics of the PPD response, or to non-specific cytotoxicity. In contrast, PPD-induced leucocyte migration inhibition was not reversed by α₁m. α₁m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, α₁m had its own leucocyte migration inhibitory effect.     

Complexes in Human Plasma between α1-Antitrypsin and IgA, and α1-Antitrypsin and Fibrinogen

Crossed immunoelectrophoretic analyses revealed that plasma contains a complex between α₁-antitrypsin¹ and IgA. Normally the complex constitutes 1–2% of these proteins. The amount varies mainly with the IgA and to some extent with the α₁-at content of the plasma. The complex is sensitive to thiol reagents and is reduced in vivo by penicillamine. The size of the complex and the amount of reduction products are compatible with the assumption of 1 IgA monomer or dimer per α₁-at molecule. An α₁-at complex with fibrinogen is also a regular plasma constituent. This complex is not susceptible to thiol reagents.     

α1-Antitrypsin deficiency in twins and parents-of-twins

Serum-trypsin-inhibitory-capacity (STIC) and alpha1-antitrypsin (AAT) genotypes were evaluated in 83 twins and 112 paired parents-of-twins. An increased prevalence (17.0--21.9%) of intermediate AAT deficiency (STIC less than 0.95 units/ml) was detected in both of these groups as compared to a prevalence of 4.1% in 1,841 healthy controls. PiS and PiZ molecular variants of AAT were also found more frequently in the twin and parent groups, but this was not statistically significant. Low levels of protease inhibition may enhance fertility and a tendency towards twinning, since proteolytic enzymes are involved in fertilization of ova by sperm and in gametogenesis. Increased fertility and twinning may be heterozygous advantages for AAT deficiency.     

Intermediate α1-antitrypsin deficiency in atopic allergy

In a population of over 200 patients with atopic bronchial disease, significantly increased frequencies of the α₁-antitrypsin Pi-types of intermediate deficiency (Pi MZ and Pi MS) were found. As far as we are aware of, this report is the first in which strictly objective criteria for patient classification with respect to atopy have been used. A possible biochemical basis for the phenomenon is presented.     

Immunological parameters and α1-antitrypsinin chronic urticaria

Serum immunoglobulins, complement and α₁-antitrypsin were assayed in forty-eight patients with chronic urticaria. Thirteen cases had chronic cold urticaria and thirty-two had chronic idiopathic urticaria. Elevated mean serum IgM was found in chronic cold urticaria. Seven patients had partial immunoglobulin deficiencies. IgE was elevated in sixteen cases of chronic idiopathic and in two with chronic cold urticaria. Eight patients had depressed serum total haemolytic complement activity. Low C3 and normal C4 serum protein concentrations in four cases suggested alternative complement pathway activation. Twenty of forty-six patients were atopic, although specific allergies responsible for the urticaria were not identified in any of them. α₁-antitrypsin levels were normal in all patients. The data suggest that the aetiology and pathogenesis of chronic urticarias in this study are heterogeneous. No evidence of abnormality of the protease inhibitor system in either chronic idiopathic or chronic cold urticaria was found.