[Effectiveness of different epidural coverage materials for preventing postoperative scar hyperplasia and dural adhesion in spine].

Objective: To analyze the effects and mechanisms of three commonly used epidural coverings, gelatin sponge, bovine Achilles tendon extract collagen and polyester urethane fiber, in preventing epidural scar adhesions after laminectomy in rats. Methods: Forty-eight adult Wistar rats were excised from L2 to L5 lamina to establish laminectomy models, and were divided into four groups with random number table according to different covering materials (12 rats in each group): blank group (group A), gelatin sponge group (absorbable, group B), polyester urethane fiber group (non-absorbable, group C) and bovine Achilles tendon extract collagen group (absorbable, group D). At 4 and 12 weeks postoperatively, the spinal tissues of the operated area were taken for gross observation (Rydell scar adhesion rating criteria) and histological observation (Nussbaum criteria); and the expression of three scar proliferation-related cytokines, basic fibroblast growth factor (bFGF), growth transforming factor ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF), were measured in the peridural tissues. The differences between the indices in each group were analyzed and compared. Results: All 48 rats survived, and gross and histological findings at 4 and 12 weeks showed no dural adhesions in the gelatin sponge and bovine Achilles tendon extract collagen groups, adhesions in the polyester urethane fiber group, and heavy adhesions and spinal cord compression in the blank control group. There were significant differences in the Rydell grade and Nussbaum histological score between the absorbable material group and the non-absorbable group (both P<0.05). Western protein blotting at 4 and 12 weeks confirmed that the expression levels of three cytokines, including bFGF, TGF-ß1, and VEGF, were lower in the absorbable material group than those in the non-absorbable group (all P<0.01). Immunofluorescence tests at 12 weeks confirmed that the expression of bFGF, TGF-ß1 and VEGF were all lower in the gelatin sponge group (9.81±0.81, 12.42±2.35, 8.63±1.76) and the bovine Achilles tendon extract collagen group (12.70±2.02, 8.23±1.03, 10.19±2.67) than those in the polyester urethane fiber group (33.94±2.03, 30.29±2.76, 25.79±1.21) (all P<0.01). Conclusions: Bovine achilles tendon extract collagen and gelatin sponge can effectively reduce the hyperplasia of scar and dural adhesions after spinal surgery.

[Analysis of perioperative blood loss of posterior lumbar interbody fusion on lumbar spondylolisthesis in patients with rheumatoid arthritis].

Objective: To compare the intraoperative blood loss, postoperative drainage and hidden blood loss (HBL) in lumbar posterior lumbar interbody fusion (PLIF) in patients with and without rheumatoid arthritis (RA), and analyze the relevant factors of HBL in RA patients. Methods: Fifty patients with RA (RA group) and 73 patients without RA (NRA group) treated in the Heze Municipal Hospital from January 2014 to April 2019 were enrolled in this study. The basic information, RA information, operation and related blood loss indicators in the two groups were compared. The intraoperative blood loss, postoperative drainage and HBL were the main results. The secondary results were operation time, preoperative and postoperative hematocrit (Hct) and hemoglobin (Hb) and their variation values, cases of anemia before and after surgery, number of new anemia after surgery, autologous blood and allogeneic blood transfusion, etc. The correlation factors of HBL in RA group were analyzed by multi-linear regression model. Results: There were 9 males and 41 females with a mean age of (62±7) years in RA group; and 11 males and 62 females with a mean age of (64±9) years in NRA group. The course of disease in RA group was (14.4±11.2) years, the most common anti-rheumatism drug (DMARDs) were single-drug and combined oral. There was no significant differences between the two groups in the number of vertebral bow screws and intervertebral fusion device. The incidence of surgical complications was comparable between the two groups. Differences between the two groups in total blood loss (TBL), intraoperative blood loss, and postoperative drainage were not statistically significant ((693±315) ml vs (630±365) ml, (454±373) ml vs (414±375) ml and (653±376) ml vs (675±400) ml, t=1.072, 0.388, -0.189, all P>0.05), while the HBL and the percentage of HBL in TBL were lower in the NRA group (t=6.157, 2.965, both P<0.05). According to the layered analysis of the number of surgical segments, the proportion of HBL and the HBL percentage of TBL in the NRA group for the long section (≥3 segments) surgery were better than those in the RA group. The Hct changing value was larger in the RA group than that in the NRA group (P=0.031). However, the difference of Hb reduction between the two groups was not statistically significant (P>0.05). There was no significant difference in anemia and exacerbation of anemia after surgery, allogeneic blood transfusion and the operation duration between the two groups (all P>0.05). A multi-linear regression analysis of HBL showed that higher RA's Steinbrocker grading, did not take DMARDS, Hb changes and infusion of allogeneic blood were independently correlated to HBL (ß=0.363, -0.272, 0.210, 1.204, all P<0.05). Conclusions: There is no difference in TBL, intraoperative blood loss, postoperative drainage and operation duration between the RA and NRA group, while HBL and the proportion of HBL in the TBL are higher in the RA group. The RA group has higher Steinbrocker rating, no DMRDs and more Hb changes.

The inhibitory effects of class I histone deacetylases on hippocampal neuroinflammatory regulation in aging mice with postoperative cognitive dysfunction.

OBJECTIVE: Neuroinflammation in the hippocampus has been determined to contribute to postoperative cognitive dysfunction (POCD) occurrence in elderly individuals. Histone deacetylases (HDACs) have been identified as important regulators of inflammation. However, the roles of different types of HDACs in POCD have never been fully explored. MATERIALS AND METHODS: POCD mouse models were established using isoflurane and validated by the Morris water maze test. The mice were pretreated with UF010 [a Class I HDAC inhibitor (HDACi)], MC1568 (a Class II HDACi) and SAHA (a Class I and II HDACi) before POCD establishment. HDAC protein levels and the activity of the NF-κB/p65, JAK/STAT and TLR/MyD88 signaling pathways in the hippocampus were investigated by Western blot (WB). The enrichment of HDACs on the promoters of genes was detected using ChIP-qPCR. RESULTS: Class I HDACs, including HDAC2 and HDAC8, and Class II HDACs, including HDAC4, HDAC7 and HDAC10, were all upregulated in the POCD group compared to the control group. Furthermore, compared to the MC1568 pretreatment group and the control group, the groups pretreated with UF010 and SAHA exhibited amelioration of the effects of anesthesia/surgery induced POCD and compromised inflammatory reactions in the hippocampus. Likewise, the NF-κB/p65, JAK/STAT and TLR/MyD88 signaling pathways were inactivated upon pretreatment with UF010 and SAHA compared to MC1568. Finally, the transcription of the genes negatively regulating these three pathways declined, and the enrichment of HDAC1, HDAC2 and HDAC8 was significantly elevated in the context of POCD. CONCLUSIONS: Class I HDACs, especially HDAC1, HDAC2 and HDAC8, play crucial roles in enhancing neuroinflammation in the hippocampus and causing POCD. Class I HDACs are potential therapeutic targets for POCD prevention and treatment via neuroinflammation inhibition.

[The role of CTLA-4 in the pathogenesis of chronic tonsillitis].

Objective:The purposes of the present study were to explore the role of CTLA-4 in the pathogenesis on development of chronic tonsillitis, and to provide new possibilities and theoretical basis for the drug therapy of chronic tonsillitis and, from soft regulation of the inflammatory immune response point of view. Method:Tonsil tissues were obtained by tonsillectomy and classified into two groups according to clinical forms of tonsillitis, CT (chronic tonsillitis) group (n=21) and TH (hypertrophy of tonsil) group (n=15). We used HE staining to observe the pathological changes in the structure of tonsil tissue in different groups. We used immunofluorescence (IF) method to determine the differences in expression of CTLA-4 between two groups and their distribution characteristics , and used Real-time fluorescent quantitative PCR technique (qRT-PCR) to detect CTLA-4 mRNA expression in the two groups. Result:①HE staining showed the pathogeny structure mainly characterized as the follicular hyperplasia and germinal center in CT group, and obvious "star" phenomenons in TH group. ②Immunofluorescence results showed that the average fluorescence intensity and density of CTLA-4 protein in CT group was significantly higher than in TH group (P<0.05), the difference was statistically significant. ③The qRT-PCR experiment results showed that the expression level of CTLA-4 mRNA in CT group were obviously higher than in HT group (t=6.294, P<0.01), and the differences were statistically significant too. Conclusion:We found CTLA-4 played an important role in the development of chronic tonsillitis disease, suggesting that the mechanism of immunosuppression may exist in the process of chronic tonsil inflammation. Tonsil lymphoid tissue immune suppression provided a new explanation for recurrent tonsillitis, and provides a possibility for the development of new drugs for the treatment of chronic tonsillitis with the exception of surgery recurrent tonsillitis, at the same time for the clinical treatment of chronic tonsillitis in addition to surgery, but the possibility of researching new drugs from the perspective of immunology.

Gynophore miRNA analysis at different developmental stages in Arachis duranensis.

Peanut is one of the most important oilseed crops in the world that provides a significant amount of lipids and protein for many people. The gynophore plays an important role in gynophore development after fertilization of the peanut ovule. MicroRNAs (miRNAs) play an important role in numerous developmental and physiological plant processes. Therefore, it is essential to analyze the miRNA sequences at different gynophore stages to explore and validate gene function. Multiple small RNAs were sequenced and collected from gynophore stages A1, A2, and A3 (5, 10, and 20 days of development, respectively) for further prediction. We obtained 266 known and 357 novel miRNAs from the three different stages. Stage A3 had the largest number of reads. Genes involved in the lignin catabolic process were identified only at stage A1. The copper ion-binding process also specifically emerged at stage A1, whereas negative regulation of biological processes occurred only in stages A2 and A3. The genes related to growth were found only at stage A3, suggesting that the gynophore may contribute to rapid development of the gynophore at this stage. Our identification and assessment of miRNAs from different gynophore stages may serve as a basis for further studies of gynophore miRNA regulation mechanisms. Some biological processes were specifically regulated at different gynophore stages indicating that miRNAs play an important role in the gynophore development.

Effective elimination of liver cancer stem-like cells by CD90 antibody targeted thermosensitive magnetoliposomes.

AIM: To investigate the use of thermosensitive magnetoliposomes (TMs) loaded with magnetic iron oxide (Fe3O4) and the anti-cancer stem cell marker CD90 (CD90@TMs) to target and kill CD90+ liver cancer stem cells (LCSCs). METHODS: The hepatocellular carcinoma cell line Huh7 was used to separate CD90+ LCSCs by magnetic-activated cell sorting. CD90@TMs was characterized and their ability to target CD90+ LCSCs was determined. Experiments were used to investigate whether CD90@TMs combined with magnetic hyperthermia could effectively eliminate CD90+ LCSCs. RESULTS: The present study demonstrated that CD90+ LCSCs with stem cells properties were successfully isolated. We also successfully prepared CD90@TMs that was almost spherical and uniform with an average diameter of 130±4.6 nm and determined that magnetic iron oxide could be incorporated and retained a superparamagnetic response. CD90@TMs showed good targeting and increased inhibition of CD90+ LCSCs in vitro and in vivo compared to TMs. CONCLUSIONS: CD90@TMs can be used for controlled and targeted delivery of anticancer drugs, which may offer a promising alternative for HCC therapy.

Increasing incidence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in Michigan and association with clinical illness.

Infection with Shiga toxin-producing Escherichia coli (STEC) by serotypes other than O157 (non-O157) have been increasingly reported in the United States. This increase in reporting is primarily due to the improvements in diagnostic tests. We analysed 1497 STEC cases reported in Michigan from 2001 to 2012. A significant increase in the number of non-O157 STEC cases was observed over time, and similar incidence rates were observed for O157 and non-O157 STEC cases in certain time periods. The odds of hospitalization was two times higher in O157 STEC cases relative to non-O157 STEC cases when adjusted for age and gender, suggesting that O157 STEC causes more severe clinical outcomes in all age groups. The use of population-based surveillance to better define trends and associations with disease severity are critical to enhance our understanding of STEC infections and improve upon current prevention and control efforts.

Tolerance for ATP-insensitive K(ATP) channels in transgenic mice.

To examine the role of sarcolemmal K(ATP) channels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac alpha-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, K(ATP) channels were very insensitive to inhibitory ATP: mean K(1/2) ([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 micromol/L in littermate control myocytes. Counterintuitively, K(ATP) channel density was approximately 4-fold lower in transgenic membrane patches than in control. This reduction of total K(ATP) conductance was confirmed in whole-cell voltage-clamp conditions, in which K(ATP) was activated by metabolic inhibition. K(ATP) conductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic beta-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac K(ATP) channels and highlight differential effects of channel activity in the electrical activity of the 2 tissues.

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential.

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.

I(Ca(TTX)) channels are distinct from those generating the classical cardiac Na(+) current.

The Na(+) current component I(Ca(TTX)) is functionally distinct from the main body of Na(+) current, I(Na). It was proposed that I(Ca(TTX)) channels are I(Na) channels that were altered by bathing media containing Ca(2+), but no, or very little, Na(+). It is known that Na(+)-free conditions are not required to demonstrate I(Ca(TTX).) We show here that Ca(2+) is also not required. Whole-cell, tetrodotoxin-blockable currents from fresh adult rat ventricular cells in 65 mm Cs(+) and no Ca(2+) were compared to those in 3 mM Ca(2+) and no Cs(+) (i.e., I(Ca(TTX))). I(Ca(TTX)) parameters were shifted to more positive voltages than those for Cs(+). The Cs(+) conductance-voltage curve slope factor (mean, -4.68 mV; range, -3.63 to -5.72 mV, eight cells) is indistinguishable from that reported for I(Ca(TTX)) (mean, -4.49 mV; range, -3.95 to -5.49 mV). Cs(+) current and I(Ca(TTX)) time courses were superimposable after accounting for the voltage shift. Inactivation time constants as functions of potential for the Cs(+) current and I(Ca(TTX)) also superimposed after voltage shifting, as did the inactivation curves. Neither of the proposed conditions for conversion of I(Na) into I(Ca(TTX)) channels is required to demonstrate I(Ca(TTX)). Moreover, we find that cardiac Na(+) (H1) channels expressed heterologously in HEK 293 cells are not converted to I(Ca(TTX)) channels by Na(+)-free, Ca(2+)-containing bathing media. The gating properties of the Na(+) current through H1 and those of Ca(2+) current through H1 are identical. All observations are consistent with two non-interconvertable Na(+) channel populations: a larger that expresses little Ca(2+) permeability and a smaller that is appreciably Ca(2+)-permeable.

Heterologous expression of the Na(+),K(+)-ATPase gamma subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore.

1. The gamma subunit is a specific component of the plasmalemmal Na(+),K(+)-ATPase. Like structurally related single-spanning membrane proteins such as cardiac phospholemman, Mat-8 and renal CHIF, large ion conductances are activated when gamma subunits are expressed in Xenopus oocytes. 2. Here we report critical properties of the gamma-activated conductance. The gamma-activated conductance showed non-selective cationic and anionic permeation, and extremely slow kinetics, with an activation time constant > 1 s following steps to -100 mV. 3. The gamma-activated conductance was inhibited by extracellular divalent ions including Ba(2+) (K(i) = 0.7 mM) and Ca(2+) (K(i) = 0.4 mM). 4. 2-Deoxyglucose (MW approximately 180), inulin (MW approximately 5000) and spermidine (MW approximately 148) efflux could occur through the gamma-activated conductance pathway, indicating a large pore diameter. In contrast, dextran-70 (MW approximately 70 000) did not pass through the gamma-activated channel, indicating an upper limit to the pore size of approximately 50 A (5 nm). 5. Similar conductances that are permeable to large molecules were activated by extreme hyperpolarization (> -150 mV) of uninjected oocytes. 6. We conclude that the Na(+),K(+)-ATPase gamma subunits activate Ca(2+)- and voltage-gated, non-selective, large diameter pores that are intrinsically present within the oocyte membrane.

ADP-ribosylation of Rab5 by ExoS of Pseudomonas aeruginosa affects endocytosis.

Pseudomonas aeruginosa exoenzyme S (ExoS) is an ADP-ribosyltransferase that modifies low-molecular-weight GTPases. Here we studied the effect of Rab5 ADP-ribosylation by ExoS on its cellular function, i.e., regulation of early endocytic events. Coculture of CHO cells with P. aeruginosa induced a marked decrease in horseradish peroxidase (HRP) uptake compared to noninfected cells, while coculture with a P. aeruginosa mutant strain that fails to produce ExoS did not lead to any change in HRP uptake. Microinjection of recombinant ExoS into Xenopus oocytes induced strong inhibition of basal HRP uptake by oocytes. Moreover, coinjection of recombinant ExoS with Rab5 abolished the typical stimulation of HRP uptake obtained after GTPase microinjection. Cytosols prepared from injected oocytes were used in an endosome-endosome fusion assay. Cytosol from ExoS-microinjected oocytes was ineffective in promoting endosome-endosome fusion. However, in these conditions, the addition of Rab5 to the assay led to fusion recovery. Finally, we found that the interaction of Rab5 with EEA1 was markedly diminished after Rab5 ADP-ribosylation by ExoS.

[Asymmetric biosynthesis of chiral styrene oxide].

Used styrene as the sole carbon and energy source, 12 strains of aerobic bacterial and two strains of fungi were screened from a series soil samples. These strains were able to convert styrene to styrene oxide. HPLC and chiral GC method were used for determination the substrate, product and e.e value of culture broth. There were obvious effect on the biomass and epoxidase activity under various conditions in the fermentation. Styrene epoxidation of the strain PS-1206 was studied in different reaction time. 35% of conversion yield and 80% e.e value of (R)-styrene oxide could been obtained at the optimum conditions.

Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.

The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit.

1. To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating channel activity, we have expressed the sulphonylurea receptor SUR1 with Kir6.2 subunits containing systematic truncations of the N- and C-termini. Up to 30 amino acids could be truncated from the N-terminus, and up to 36 amino acids from the C-terminus without loss of functional channels in co-expression with SUR1. Furthermore, Kir6.2DeltaC25 and Kir6. 2DeltaC36 subunits expressed functional channels in the absence of SUR1. 2. In co-expression with SUR1, N-terminal truncations increased Ki,ATP ([ATP] causing half-maximal inhibition of channel activity) by as much as 10-fold, accompanied by an increase in the ATP-insensitive open probability, whereas the C-terminal truncations did not affect the ATP sensitivity of co-expressed channels. 3. A mutation in the near C-terminal region, K185Q, reduced ATP sensitivity of co-expressed channels by approximately 30-fold, and on the Kir6.2DeltaN2-30 background, this mutation decreased ATP sensitivity of co-expressed channels by approximately 400-fold. 4. Each of these mutations also reduced the sensitivity to inhibition by ADP, AMP and adenosine tetraphosphate. 5. The results can be quantitatively explained by assuming that the N-terminal deletions stabilize the ATP-independent open state, whereas the Kir6.2K185Q mutation may alter the stability of ATP binding. These two effects are energetically additive, causing the large reduction of ATP sensitivity in the double mutant channels.

The gamma subunit of the Na,K-ATPase induces cation channel activity.

The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.

Inhibition of an inward rectifier potassium channel (Kir2.3) by G-protein betagamma subunits.

The molecular basis of G-protein inhibition of inward rectifier K+ currents was examined by co-expression of G-proteins and cloned Kir2 channel subunits in Xenopus oocytes. Channels encoded by Kir2.3 (HRK1/HIR/BIRK2/BIR11) were completely suppressed by co-expression with G-protein betagamma subunits, whereas channels encoded by Kir2. 1 (IRK1), which shares 60% amino acid identity with Kir2.3, were unaffected. Co-expression of Galphai1 and Galphaq subunits also partially suppressed Kir2.3 currents, but Galphat, Galphas, and a constitutively active mutant of Galphail (Q204L) were ineffective. Gbetagamma and Kir2.3 subunits were co-immunoprecipitated using an anti-Kir2.3 antibody. Direct binding of G-protein betagamma subunits to fusion proteins containing Kir2.3 N terminus, but not to fusion proteins containing Kir2.1 N terminus, was also demonstrated. The results are consistent with suppression of Kir2.3 currents resulting from a direct protein-protein interaction between the channel and G-protein betagamma subunits. When Kir2.1 and Kir2.3 subunits were coexpressed, the G-protein inhibitory phenotype of Kir2.3 was dominant, suggesting that co-expression of Kir2.3 with other Kir subunits might give rise to novel G-protein-inhibitable inward rectifier currents.

Depletion of intracellular polyamines relieves inward rectification of potassium channels.

Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches.

Spermidine release from xenopus oocytes. Electrodiffusion through a membrane channel.

The mechanism of spermidine release from Xenopus oocytes was examined by measuring release of radioactive [3H]spermidine under different ionic conditions, and under voltage-clamp. In normal solution (2 mM K+), the efflux rate is less than 1% per hour, and is stimulated approximately 2-fold by inclusion of Ca2+ (1 mm) in the incubation medium. Spermidine efflux is stimulated approximately 10-fold in high [K+] (KD98) solution. In KD98 solution, efflux is strongly inhibited by divalent cations (Ki for Ba2+ block of spermidine efflux is approximately 0.1 mM), but not by tetraethylammonium ions or verapamil. Spermidine efflux rates were not different between control oocytes and those expressing HRK1 inward rectifier K+ (Kir) channels. When the membrane potential was clamped, either by changing external [K+] in oocytes expressing HRK1, or by 2-microelectrode voltage-clamp, spermidine efflux was shown to be strongly dependent on voltage, as expected for a simple electrodiffusive process, where spermidine3+ is the effluxing species. This result argues against spermidine diffusing out as an uncharged species, or in exchange for similarly charged counterions. These results are the first conclusive demonstration of a simple electrodiffusive pathway for spermidine efflux from cells.