Impaired Antibody Responses but Normal Proliferation of Specific CD4+ T Cells in Mice Lacking Complement Receptors 1 and 2

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4⁺ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro , B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2^−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2^−/− and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2^−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2^−/− mice cannot be explained by a lack of appropriate induction of T cell help.     

The kinin B1 receptor contributes to the cardioprotective effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in mice

Recent studies have shown that inhibition of angiotensin-converting enzyme (ACE) or angiotensin II receptors causes upregulation of the B₁ receptor (B₁R). Here we tested the hypothesis that activation of the B₁R partly contributes to the cardiac beneficial effect of ACE inhibitor (ACEi) and angiotensin II receptor blockers (ARB). B₁R knockout mice ( B₁R^−/− ) and C57Bl/6J (wild-type control animals, WT) were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. Three weeks after MI, each strain of mice was treated with vehicle, ACEi (ramipril, 2.5 mg kg⁻¹ day⁻¹ in drinking water) or ARB (valsartan, 40 mg kg⁻¹ day⁻¹ in drinking water) for 5 weeks. We found that: (1) compared with WT mice, B₁R^−/− mice that underwent sham surgery had slightly but significantly increased left ventricular (LV) diastolic dimension, LV mass and myocyte size, whereas systolic blood pressure, cardiac function and collagen deposition did not differ between strains; (2) MI leads to LV hypertrophy, chamber dilatation and dysfunction similarly in both WT and B₁R^−/− mice; and (3) ACEi and ARB improved cardiac function and remodelling in both strains; however, these benefits were significantly diminished in B₁R^−/− mice. Our data suggest that kinins, acting via the B₁R, participate in the cardioprotective effects of ACEi and ARB.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

TLR9 Activation Increases TAP-Independent Vesicular MHC Class I Processing In vivo

Cross-presentation of soluble protein antigens on major histocompatibility complex (MHC) class I by dendritic cells (DC) can occur in vesicular, endolysosomal compartments and be either dependent or independent of TAP peptide transporters. Here we investigate if an immunostimulatory CpG oligodeoxynucleotide can increase the activity in a TAP-independent endolysosomal vesicular pathway (el-VP) in vivo as we have earlier found in in vitro cultured DC. We use the in vivo response of CFSE labelled OT-1 T cells, transgenic for a T-cell receptor (TCR) that recognizes an ovalbumin (OVA)-derived peptide (SIINFEKL) presented by H-2K^b, transferred into TAP1^−/− mice, as a functional read-out for activity in the el-VP. We have found a poor OT-1 T-cell response to soluble OVA which, however, could be strongly enhanced by the simultaneous administration of CpG. This increased responsiveness required both the endolysosomal cathepsin S (CatS) and Toll like receptor (TLR)9, the CpG receptor, both of which are present in the el-VP. Confocal microscopy demonstrated a co-localization of H-2K^b/SIINFEKL and the endolysosomal marker LAMP1 in CD11c positive DC which was markedly increased by CpG administration. No complexes were found in the ER and cis-Golgi compartments in TAP1^−/− mice, indicating the lack of classical MHC-I processing. In DC isolated from CatS^−/− mice the opposite was found, complexes were present in the ER but not in the el-VP. We conclude that in vivo activation of TLR9 by CpG increases the efficiency of TAP independent el-VP and that this might contribute to the potent adjuvant activity of this type of compound. The cellular mechanisms remain to be established.     

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

Local metabolite accumulation augments passive muscle stretch-induced modulation of carotid–cardiac but not carotid–vasomotor baroreflex sensitivity in man

We examined the effects of muscle mechanoreflex stimulation by passive calf muscle stretch, at rest and during concurrent muscle metaboreflex activation, on carotid baroreflex (CBR) sensitivity. Twelve subjects either performed 1.5 min one-legged isometric plantarflexion at 50% maximal voluntary contraction with their right or left calf [two ischaemic exercise (IE) trials, IER and IEL] or rested for 1.5 min [two ischaemic control (IC) trials, ICR and ICL]. Following exercise, blood pressure elevation was partly maintained by local circulatory occlusion (CO). 3.5 min of CO was followed by 3 min of CO with passive stretch (STR-CO) of the right calf in all trials. Carotid baroreflex function was assessed using rapid pulses of neck pressure from +40 to −80 mmHg. In all IC trials, stretch did not alter maximal gain of carotid–cardiac (CBR–HR) and carotid–vasomotor (CBR–MAP) baroreflex function curves. The CBR–HR curve was reset without change in maximal gain during STR-CO in the IEL trial. However, during the IER trial maximal gain of the CBR–HR curve was smaller than in all other trials (−0.34 ± 0.04 beats min⁻¹ mmHg⁻¹ in IER versus −0.76 ± 0.20, −0.94 ± 0.14 and −0.66 ± 0.18 beats min⁻¹ mmHg⁻¹ in ICR, IEL and ICL, respectively), and significantly smaller than in IEL ( P < 0.05). The CBR–MAP curves were reset from CO values by STR-CO in the IEL and IER trials with no changes in maximal gain. These results suggest that metabolite sensitization of stretch-sensitive muscle mechanoreceptive afferents modulates baroreflex control of heart rate but not blood pressure.     

Apolipoprotein A-IV is involved in detection of lipid in the rat intestine

Long chain triglyceride (>C12) in the intestinal lumen potently inhibits gastric emptying and acid secretion via the vagal afferent pathway. While the mechanism of inhibition involves the formation of chylomicrons, the essential role of the apolipoprotein apo A-IV is unclear. Using apo A-IV^−/− mice, we tested the hypothesis that inhibition of gastric emptying and gastric acid secretion in response to dietary lipid is dependent upon apo A-IV. As measured by nuclear scintigraphy in awake mice, gastric emptying of an ingested whole-egg meal was significantly faster in apo A-IV^−/− knockout versus A-IV^+/+ controls (34 ± 1 versus 54 ± 3 min, P < 0.0001). In anaesthetized A-IV^+/+ mice, meal-stimulated gastric acid secretion was 59% inhibited by intestinal lipid infusion; this was abolished in apo A-IV^−/− mice. Oral gavage of lipid in awake mice activated neurones throughout the nucleus of the solitary tract (NTS) in A-IV^+/+ mice, measured by immunohistochemical localization of Fos protein expression. However, in the mid region of the NTS (bregma −7.32 to −7.76 mm), Fos expression in response to intestinal lipid was significantly decreased by 50% in apo A-IV^−/− mice compared to A-IV^+/+ controls. We conclude that activation of the vagal afferent pathway and inhibition of gastric function in response to dietary lipid is partly dependent upon apo A-IV.     

IC31®, a Two-Component Novel Adjuvant Mixed with a Conjugate Vaccine Enhances Protective Immunity against Pneumococcal Disease in Neonatal Mice

IC31^® is a novel adjuvant which combines the immunostimulatory effects of an 11-mer antibacterial peptide (KLKL₅KLK) and a synthetic oligodeoxynucleotide (ODN1a) which is a Toll-like receptor 9 agonist without containing cytosine phosphate guanine (CpG) motifs. The effects of IC31^® on neonatal immune response to vaccination have not been reported. Neonatal mice were immunized once or twice with a Streptococcus pneumoniae serotype 1 polysaccharide conjugate containing Tetanus Toxoid (Pnc1-TT) carrier protein, with or without IC31^® or CpG-ODN. IC31^® significantly enhanced IgG1, IgG2a and IgG2b antibodies (Ab) to the serotype 1 polysaccharide. One dose of Pnc1-TT and low dose IC31^® elicited high Ab levels that protected the neonatal mice completely from bacteraemia and significantly reduced lung infection following i.n. challenge with serotype 1 pneumococcal strain. One-sixth of an adult murine dose of IC31^® was sufficient and optimal for induction of protective immunity in neonatal mice. Two doses of Pnc1-TT with or without adjuvants protected the neonatal mice completely, but more rapid Ab response was observed when IC31^® was given with the Pnc1-TT. IC31^® is a promising new adjuvant for neonatal vaccinations, rapidly enhancing protective humoral responses when combined with Pnc1-TT.     

Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.     

Proton modulation of ion channels in isolated horizontal cells of the goldfish retina

Transient changes in extracellular pH (pH_o) occur in the retina and may have profound effects on neurotransmission and visual processing due to the pH sensitivity of ion channels. The present study characterized the effects of acidification on the activity of membrane ion channels in isolated horizontal cells (HCs) of the goldfish retina using whole-cell patch-clamp recording. Currents recorded from HCs were characterized by prominent inward rectification at potentials negative to −80 mV, a negative slope conductance between −70 and −40 mV, a sustained inward current, and outward rectification positive to 40 mV. Inward currents were identified as those of inward rectifier K⁺ (Kir) channels and Ca²⁺ channels by their sensitivity to 10 m m Cs⁺ or 20 μ m Cd²⁺, respectively. Both of these currents were reduced when pH_o decreased from 7.8 to 6.8. Glutamate (1 m m )-activated currents were also identified, as were hemichannel currents that were enhanced by removal of extracellular Ca²⁺ and application of 1 m m quinidine. Both glutamate-activated and hemichannel currents were suppressed by a similar reduction of pH_o. When all of these H⁺-inhibited currents were blocked, a small, sustained inward current at −60 mV increased following a decrease in pH_o from 7.8 to 6.8. In addition, slope conductance between −70 and −20 mV increased during this acidification. Suppression of this H⁺-activated current by removal of extracellular Na⁺, and an extrapolated E _rev near E _Na, indicated that this current was carried predominantly by Na⁺ ions.     

Temperature-sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes

TREK channels belong to the superfamily of two-pore-domain K⁺ channels and are activated by membrane stretch, arachidonic acid, volatile anaesthetics and heat. TREK-1 is highly expressed in the atrium of the adult heart. In this study, we investigated the role of TREK-1 and TREK-2 channels in regulating the resting membrane potential (RMP) of isolated chicken embryonic cardiac myocytes. At room temperature, the average RMP of embryonic day (ED) 11 atrial myocytes was −22 ± 2 mV. Raising the temperature to 35°C hyperpolarized the membrane to −69 ± 2 mV and activated a large outwardly rectifying K⁺ current that was relatively insensitive to conventional K⁺ channel inhibitors (TEA, 4-AP and Ba²⁺) but completely inhibited by tetracaine (200 μ m ), an inhibitor of TREK channels. The heat-induced hyperpolarization was mimicked by 10 μ m arachidonic acid, an agonist of TREK channels. There was little or no inwardly rectifying K⁺ current ( I _K1) in the ED11 atrial cells. In marked contrast, ED11 ventricular myocytes exhibited a normal RMP (−86.1 ± 3.4 mV) and substantial I _K1, but no temperature- or tetracaine-sensitive K⁺ currents. Both RT-PCR and real-time PCR further demonstrated that TREK-1 and TREK-2 are highly and almost equally expressed in ED11 atrium but much less expressed in ED11 ventricle. In addition, immunofluorescence demonstrated TREK-1 protein in the membrane of atrial myocytes. These data indicate the presence and function of TREK-1 and TREK-2 in the embryonic atrium. Moreover, we demonstrate that TREK-like currents have an essential role in determining membrane potential in embryonic atrial myocytes, where I _K1 is absent.     

Mast cell-cholinergic nerve interaction in mouse airways

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 μg ml⁻¹) caused histamine release (∼70% total tissue content), and smooth muscle contraction that was rapid in onset and short-lived ( t _1/2 < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 μ m -ketanserin (5-HT₂ receptor antagonist) and strongly inhibited by atropine (1 μ m ). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient ( sash −/−) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient ( sash −/−) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT₂ receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.     

Differences in interaction between immune complexes and complement receptors in serum and cerebrospinal fluid

Antigen—antibody complexes (Ag—Ab), prepared from ¹²⁵I-radiolabelled bovine serum albumin (BSA) and guinea-pig antibody, were (1) pre-incubated at 37°C for 30 min with serum or cerebrospinal fluid (CSF) in different proportions and then reacted with cells, (2) incubated at 37°C directly with cells suspended in serum or CSF for different time periods, or (3) bound to cells (following incubation with serum in optimal proportions) and the cell-bound immune complexes (IC) incubated with serum or CSF at 37°C for different time periods. When Ag—Ab were pre-incubated with serum or CSF and reacted with unfractionated blood cells or mononuclear cells, binding decreased as serum to Ag—Ab proportion was increased above 1:16, but increased as CSF to Ag—Ab proportion was increased. When serum diluted 200-fold (to approximate the protein concentration of CSF) was used in place of undiluted serum, serum-mediated binding paralleled CSF-mediated binding. Inactivation of serum, CSF, and 1:200 serum in different ways and substitution of human red blood cells (RBC) (known to possess C3b receptors) or sheep RBC (known not to possess C3b receptors) demonstrated that binding was to C3b receptors. Addition of CSF to serum did not alter serum-mediated binding. When Ag—Ab were incubated directly with unfractionated blood cells suspended in serum or CSF, binding increased rapidly in serum, reaching a maximum within 2—4 min, and IC then rapidly dissociated, whereas binding increased gradually in CSF and IC remained associated with cells. When serum diluted approximately 100-fold was used in place of undiluted serum, kinetics of serum-mediated interaction approached that of CSF-mediated interaction. When IC were bound to Raji cells or human RBC and the cell-bound IC incubated in serum or CSF, > 85% of IC dissociated in serum after 30 min, but no dissociation occurred in CSF. Dilution of serum > 1:16 and > 1:64 abolished dissociation from the two cell types, respectively. These results indicate that CSF mediates binding of IC to complement receptors on cells but lacks the activities of serum which convert IC into a non-binding state.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

Inefficient Binding of IgM Immune Complexes to Erythrocyte C3b–C4b Receptors (CR1) and Weak Incorporation of C3b–iC3b into the Complexes

The binding of soluble complement-reacted IgM immune complexes (IC) to erythrocyte (E) C3b–C4b receptors (CRI) and the incorporation of C3b–iC3b into solid phase IgM-IC was investigated. The optimal binding of liquid phase IgM-IC to E-CRI was obtained with IC formed at moderate antibody excess, but the binding was low (2–3%) when compared to the binding of the corresponding IgG-IC (50–60%). Solid phase IC were prepared by coming microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37°C. The binding of C3b–iC3b was determined by use of biotinylated F(ab')₂ antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b–iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching a maximum after about 2 h at 37°C. The binding of C3b–iC3b to the IgM-IC was remarkably inefficient when compared to the incorporation into IgG-IC reacted with the same amounts of BSA-precipitating antibody.     

Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current

Potassium channels play an important role in controlling the excitability of urinary bladder smooth muscle (UBSM). Here we describe the biophysical, pharmacological and molecular properties of the mouse UBSM voltage-gated K⁺ current ( I _{K ( V)}). The I _{K ( V)} activated, deactivated and inactivated slowly with time constants of 29.9 ms at +30 mV, 131 ms at −40 mV and 3.4 s at +20 mV. The midpoints of steady-state activation and inactivation curves were 1.1 mV and −61.4 mV, respectively. These properties suggest that I _{K ( V)} plays a role in regulating the resting membrane potential and contributes to the repolarization and after-hyperpolarization phases of action potentials. The I _{K ( V)} was blocked by tetraethylammonium ions with an IC₅₀ of 5.2 m m and was unaffected by 1 m m 4-aminopyridine. RT-PCR for voltage-gated K⁺ channel (K_V) subunits revealed the expression of Kv2.1, Kv5.1, Kv6.1, Kv6.2 and Kv6.3 in isolated UBSM myocytes. A comparison of the biophysical properties of UBSM I _{K ( V)} with those reported for Kv2.1 and Kv5.1 and/or Kv6 heteromultimeric channels demonstrated a marked similarity. We propose that heteromultimeric channel complexes composed of Kv2.1 and Kv5.1 and/or Kv6 subunits form the molecular basis of the mouse UBSM I _{K ( V)}.     

Establishment and Characterization of RAG-2 Deficient Non-Obese Diabetic Mice

The authors have established a new immunodeficient mouse strain on the genetic background of the diabetes prone non-obese diabetic (NOD) mouse. A deletion mutant of the RAG-2 gene was back crossed 10 generations onto the NOD/Bom strain background. The homozygous NOD^{rag−2−/−} mice lack functionally mature B and T lymphocytes and do not develop insulitis or diabetes throughout life. In contrast, heterozygous NOD^rag−2+/− develop both insulitis and diabetes with an incidence similar to the wild type NOD mice. In transfer experiments, spleen cells from diabetic NOD donors were found to transfer disease to NOD^{rag−2−/−} recipients similar to what has been previously observed in transfer to irradiated NOD recipients or to immunodeficient NOD-scid/scid mice. While resembling the recently established NOD-scid/scid mice in many respects, the NOD^{rag−2−/−} mice represents an advantageous model for reconstitution of the pathogenesis of murine IDDM as it does not produce any endogenous, mature T or B lymphocytes.     

Baroreflex-induced sympathetic activation does not alter cerebrovascular CO2 responsiveness in humans

We investigated the effect of baroreflex-induced sympathetic activation, produced by lower body negative pressure (LBNP) at −40 mmHg, on cerebrovascular responsiveness to hyper- and hypocapnia in healthy humans. Transcranial Doppler ultrasound was used to measure blood flow velocity (CFV) in the middle cerebral artery during variations in end-tidal carbon dioxide pressure ( P _ET,CO2) of +10, +5, 0, −5, and −10 mmHg relative to eupnoea. The slopes of the linear relationships between P _ET,CO2 and CFV were computed separately for hyper- and hypocapnia during the LBNP and no-LBNP conditions. LBNP decreased pulse pressure, but did not change mean arterial pressure. LBNP evoked an increase in ventilation that resulted in a 9 ± 2 mmHg decrease in P _ET,CO2, which was corrected by CO₂ supplementation of the inspired air. LBNP did not affect cerebrovascular CO₂ response slopes during steady-state hypercapnia (3.14 ± 0.24 vs. 2.96 ± 0.26 cm s⁻¹ mmHg⁻¹) or hypocapnia (1.31 ± 0.18 vs. 1.32 ± 0.19 cm s⁻¹ mmHg⁻¹), or the CFV responses to voluntary apnoea (+51 ± 19 vs. +50 ± 18 %). Thus, cerebrovascular CO₂ responsiveness was not altered by baroreflex-induced sympathetic activation. Our data challenge the concept that sympathetic activation restrains cerebrovascular responses to alterations in CO₂ pressure.