Alternate-day fat diet and exenatide modulate the brain leptin JAK2/STAT3/SOCS3 pathway in a fat diet-induced obesity and insulin resistance mouse model.

Introduction: Obesity is one of the most burdensome health problems and is closely linked to leptin resistance. The study examined whether an alternate-day high-fat diet (ADF) and/or GLP-1 agonist (exenatide) modulate brain leptin resistance caused by a high-fat diet (HFD). Material and methods: Sixty adult male mice were divided into 6 groups: (i) normal palatable diet (NPD), (ii) exenatide control (NPD received exenatide) (iii) HFD, (iv) ADF treated, (v) exenatide treated, (vi) ADF and exenatide treated. All animal groups were fed a HFD for 8 weeks, before they received treatment (ADF and/or exenatide) for 8 additional weeks. Body weight was assessed at the start and at the end of the experiment. Lipid profile, brain leptin and its receptor expression with the leptin-sensitive pathway, JAK2/STAT3/SOCS3/PTP1B, fasting blood glucose (FBG), serum insulin, liver metabolic handling via its regulators IRS1/PI3K/GLUT4 for hyperinsulinemia/obesity-induced PDK3/NAFLD2 modification, and liver enzymes were determined at the end of the experiment. Results: ADF and exenatide reduced body weight and FBG in HFD-obese mice (p < 0.05). The combined ADF and exenatide regimen enhanced the brain anorexic leptin/JAK2/STAT3 and attenuated the SOCS3/PTP1B pathway (p < 0.05). The ADF/exenatide anorexigenic brain effect also modulated liver glucose via IRS1/PI3K/GLUT4 expression (p < 0.05), attenuating NAFLD2 and PDK3 expression (p < 0.05). Liver enzymes and the histopathological profile confirmed the improvement. Conclusions: In HFD caloric consumption, a combination of ADF and GLP-1 agonist enhances the brain leptin anorexigenic effect with the improvement of the metabolic sequelae of hyperinsulinemia, hyperlipidemia and liver steatosis.

Upregulation of antioxidant nuclear factor erythroid 2-related factor 2 and its dependent genes associated with enhancing renal ischemic preconditioning renoprotection using levosimendan and cilostazol in an ischemia/reperfusion rat model.

INTRODUCTION: Ischemic preconditioning (Ipre) provides protection against renal ischemia-reperfusion (I/R) injury with its associated remote organ damage. This study examined the enhancing protective effect of Ipre with levosimendan or cilostazol in I/R-induced kidney and lung injury in a rat model. MATERIAL AND METHODS: Rats were divided into: sham-operated, I/R control, Ipre control, I/R + cilostazol or levosimendan and Ipre + cilostazol or levosimendan. Drugs were given 30 min before left renal I/R or 4 cycles of Ipre just before renal ischemia. RESULTS: The Ipre combined with the implemented drugs enhanced physiological antioxidant defense genes including renal nuclear factor erythroid 2-related factor 2 (Nrf2) and its dependent genes heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase-1 (NQO-1) and improved malondialdehyde and superoxide dismutase renal tissue levels. The combined effect improved I/R consequences for blood urea, creatinine, and creatinine clearance and improved blood oxygenation and metabolic acidosis. Moreover, the combination improved the renal soluble intercellular adhesion molecule (ICAM), tumor necrosis factor α (TNF-α) and interlukin-6 (IL-6) with histopathological improvement of tubular necrosis with a decrease in the apoptotic marker caspase-3 and an increase in the anti-apoptotic Bcl-2 expression. CONCLUSIONS: Cilostazol or levosimendan potentiates the renoprotective effect of Ipre against renal I/R injury, associated with upregulation of antioxidant genes Nrf2, HO-1, and NOQ-1 expression.

Blocking angiotensin 2 receptor attenuates diabetic nephropathy via mitigating ANGPTL2/TL4/NF-κB expression.

BACKGROUND: Diabetic nephropathy (DN) is a consequence of diabetes mellitus (DM) and is associated with early changes in renal angiotensin II (ANG II). These changes were evaluated using ANG II blocker valsartan early from week two of diabetes (experiment I, renoprotective) and late from week nine of diabetes (experiment II, renotherapeutic) to the end of both experiments at week twelve. METHODS AND RESULTS: In both experiments, adult male Wister rats were divided into (i) vehicle group; (ii) valsartan received oral 30 mg/Kg/day; (iii) diabetic received single 50 mg/Kg intraperitoneal streptozotocin injection; (iv) renoprotection, diabetic rats received valsartan treated in experiments I and II. DM effects on urine albumin excretion, blood pressure, and renal ANG II were measured. Urinary nephrin, kidney injury molecule-1 (KIM-1), renal angiopoietin-like protein 2 (ANGPTL2), and toll-like receptor 4 (TLR 4) mRNA expression were tested. DM-initiated fibrotic markers integrin, α-smooth muscle actin expression, and collagen IV and apoptotic protein caspase 3 were tested. DM induced early changes starting from week four in the tested variables. At week twelve, in both experiments, valsartan intervention showed a significant reduction in ANG II, ANGPTL2, TLR 4 and integrin expression and improvement in albuminuria, blood pressure, urinary biomarkers, fibrotic and apoptotic markers. CONCLUSIONS: Changes leading to DN starts early in the disease course and ANG II reduction decreased the expression of ANGPTL2 and integrin which preserve the glomerular barrier. Blocking ANG II was able to decrease TLR 4 and inflammatory cytokines leading to decreasing DN.

Clopidogrel or prasugrel reduces mortality and lessens cardiovascular damage from acute myocardial infarction in hypercholesterolemic male rats.

AIMS: Hypercholesterolemia is a hazard for increasing susceptibility of the heart to myocardial infarction (MI) by inducing platelet hyperaggregability. Clopidogrel and prasugrel have documented cardioprotective effects in clinical studies. Herein, we investigated whether clopidogrel and prasugrel could protect against isoproterenol-induced acute MI (A-MI) under hypercholesterolemic conditions in rats. MAIN METHODS: Dietary hypercholesterolemic rats were subjected to acute doses of isoproterenol. Serum lipids, inflammatory markers, aortic endothelin1 and endothelial nitric oxide synthase (eNOS) mRNAs expression and immunexpression of BCL2 were determined. KEY FINDINGS: Hypercholesterolemic rats showed infiltration of inflammatory cells and reduction in aortic wall thickness, deposition of fibrous tissue between cardiac muscle fibers. Protective doses of prasugrel or clopidogrel for 28 days before A-MI increased survival, amended the ECG parameters -including ST segment elevation- and improved the histopathological picture in hypercholesterolemic rats. This was coupled with reductions in platelet aggregation, creatine kinase-MB activity, endothelin 1, systemic inflammation and cardiac lipid peroxidation and increment in aortic eNOS expression. Clopidogrel and prasugrel groups showed enhanced BCL2 expression in cardiac fibers and aortic wall. SIGNIFICANCE: Prasugrel and clopidogrel protected against A-MI via anti-aggregatory and anti-inflammatory effects. These results add to the value of these drugs in correcting cardiovascular dysfunction in patients vulnerable to A-MI after confirmation by appropriate human studies.

Neuroprotective effect of levetiracetam in mouse diabetic retinopathy: Effect on glucose transporter-1 and GAP43 expression.

AIMS: Retinopathy is a neurodegenerative complication associating diabetes mellitus. Diabetic retinopathy (DR) is the primary reason of visual loss during early adulthood. DR has a complicated multifactorial pathophysiology initiated by hyperglycaemia-induced ischaemic neurodegenerative retinal changes, followed by vision-threatening consequences. The main therapeutic modalities for DR involve invasive delivery of intravitreal antiangiogenic agents as well as surgical interventions. The current work aimed to explore the potential anti-inflammatory and retinal neuroprotective effects of levetiracetam. MAIN METHODS: This study was performed on alloxan-induced diabetes in mice (n: 21). After 10 weeks, a group of diabetic animals (n: 7) was treated with levetiracetam (25 mg/kg) for six weeks. Retinal tissues were dissected and paraffin-fixed for examination using (1) morphometric analysis with haematoxylin and eosin (HE), (2) immunohistochemistry (GLUT1, GFAP and GAP43), and (3) RT-PCR-detected expression of retinal inflammatory and apoptotic mediators (TNF-α, IL6, iNOS, NF-κB and Tp53). KEY FINDINGS: Diabetic mice developed disorganized and debilitated retinal layers with upregulation of the gliosis marker GFAP and downregulation of the neuronal plasticity marker GAP43. Additionally, diabetic retinae showed increased transcription of NF-κB, TNF-α, IL6, iNOS and Tp53. Levetiracetam-treated mice showed downregulation of retinal GLUT1 with relief and regression of retinal inflammation and improved retinal structural organization. SIGNIFICANCE: Levetiracetam may represent a potential neuroprotective agent in DR. The data presented herein supported an anti-inflammatory role of levetiracetam. However, further clinical studies may be warranted to confirm the effectiveness and safety of levetiracetam in DR patients.

Protective effect of mirtazapine versus ginger against cisplatin-induced testicular damage in adult male albino rats.

Cisplatin (CP) is a chemotherapy medication used to treat different types of organs cancers. It has damaging effects on testes. Mirtazapine is an antidepressant, which is used primarily in the treatment of depression and other anxiety disorders. Ginger is a naturally growing plant with antioxidant properties. Thirty-six adult male albino rats, subdivided into six groups (six animals each) received treatment for 30 days. Group I (control) received saline solution orally; group II received mirtazapine (20 mg/kg). Group III received ginger (200 mg/kg/day), group IV received CP (7 mg/kg) IP single dose, at day 23rd, group V received mirtazapine (200 mg/day) orally till day 23rd, CP (7 mg/kg) IP at day 23rd, mirtazapine till day 30th, group VI received ginger (200 mg/Kg/day) orally till day 23rd, CP (7 mg/kg) IP at day 23rd, and then ginger at the previous dose till day 30th. This study examined the microscopic changes associated with CP and the possible testicular protective role of mirtazapine versus ginger of adult male rats. Mirtazapine and ginger resulted in cellular protection of testicular tissue as evident from microscopic changes including Sertoli cells, spermatogonia, and Leydig cells. Ginger showed to have a more protective effect than mirtazapine on testicular tissue against CP treatment.

Angiotensin blockade attenuates diabetic nephropathy in hypogonadal adult male rats.

This study examined the effect of the aromatase inhibitor letrozole (0.5 mg/kg) alone or in combination with the angiotensin-receptor blocker valsartan (30 mg/kg) against streptozocin-induced diabetic nephropathy (DN) in hypogonadal (HG) rats for 12 weeks. First, we tested the HG effect on hormone levels, inflammatory cytokines, and oxidative stress in nondiabetic (ND) and diabetic (D) rats. HG was induced with the luteinizing hormone-releasing hormone antagonist cetrorelix (0.71 mg/kg). Diabetes enhanced hormonal hypogonadism and increased inflammation and oxidative stress. Next, experiments examined the effect of early letrozole and valsartan intervention on DN in HG rats. HG-ND and HG-D rats were treated with letrozole alone or in combination with valsartan. HG-D rats developed proteinuria and had increased blood urea nitrogen and creatinine, and histopathological evidence of renal injury, including glomerular hypertrophy and mesangial expansion. Valsartan alone or in combination with letrozole reduced proteinuria, improved renal functions, and reduced diabetes-induced renal angiotensin II. Both agents ameliorated nuclear factor kappa light chain enhancer of activated B cells, interleukin 1ß, interleukin 6, and tumor necrosis factor alpha levels. The combination decreased superoxide dismutase, malondialdehyde, and glutathione peroxidase levels, and prevented glomerular hypertrophy. In HG-D rats, valsartan reduced renal collagen IV and transforming growth factor-beta 1, especially when the testosterone level was corrected by letrozole. Thus, normalizing testosterone and inhibiting renal angiotensin II have a renoprotective effect against DN in HG male rats.

Cardioprotective and Anti-Aggregatory Effects of Levosimendan on Isoproterenol-Induced Myocardial Injury in High-Fat-Fed Rats Involves Modulation of PI3K/Akt/mTOR Signaling Pathway and Inhibition of Apoptosis: Comparison to Cilostazol.

Hyperlipidemia and hypercoagulability states are linked with the increased risks of myocardial infarction (MI). Levosimendan has vasorelaxant and anti-aggregatory properties. The present study evaluated the anti-aggregatory and cardioprotective effects of levosimendan versus cilostazol in high-fat diet (HFD)-fed rats subjected to isoproterenol-induced MI. Rats were assigned to normal, HFD, HFD + isoproterenol, HFD + isoproterenol + cilostazol, and HFD + isoproterenol + levosimendan. The present study investigated the anti-aggregatory effect of both levosimendan and cilostazol and revealed that both drugs attenuated the severity of platelet aggregation. Moreover, both levosimendan and cilostazol revealed effectiveness in attenuating the severity of HFD/isoproterenol-induced myocardial injury as revealed by electrocardiogram signs, apoptotic markers, and histopathological score via counteracting the oxidative stress burden, increments in the expression of inflammatory mediators, and modulating nuclear factor kappa-B (NF-κB) and phosphatidylinositide 3-kinases (PI3K)/protein kinase B (Akt)/ mechanistic target of rapamycin (mTOR) pathway. It was obvious that levosimendan offered more cardioprotective properties than cilostazol. The study showed the relations between hyperlipedemia, hyperaggregability state, and myocardial injury with the modulation of NF-κB and PI3K/Akt/mTOR pathway.

Oxidative stress burden inhibits spermatogenesis in adult male rats: testosterone protective effect.

In this study, we aimed to investigate the protective effects of androgens, using letrozole (LET; an aromatase inhibitor), grape seed extract (GSE; a naturally occurring aromatase inhibitor and antioxidant), and testosterone propionate (Tp), against methotrexate (MTX)-induced testicular toxicity in adult male rats. MTX has been shown to induce oxidative stress and exhibit antiproliferative effects in the testes. Adult male rats received oral saline gavage (control group with no treatment), the potential protective agents (LET, GSE, or Tp) alone, MTX alone, or a combination of one of the potential protective agents and MTX. The testicular levels of oxidative stress markers and cytokines (tumor necrosis factor-α and interleukin-1ß) were measured. Spermatogenesis and sperm viability were microscopically evaluated. Administration of LET and GSE 7 days before MTX improved spermatogenesis and sperm viability, as well as reduced the levels of oxidative stress markers and cellular cytokines. Exogenous testosterone exhibited anti-inflammatory and antioxidant activities, similar to GSE and LET. We also showed that enhancing the endogenous androgenic activity by LET and GSE protected spermatogenesis against MTX-induced testicular toxicity via reduction of inflammation and oxidative stress in the testes. Our data suggest that testosterone protected spermatogenesis owing to its antioxidant and anti-inflammatory properties.

5-HT7 receptor antagonism (SB-269970) attenuates bleomycin-induced pulmonary fibrosis in rats via downregulating oxidative burden and inflammatory cascades and ameliorating collagen deposition: Comparison to terguride.

The neurotransmitter 5-hydroxytryptamine (5-HT) is involved in regulation of local tissue inflammation and repair through a set of receptors (5-HT1-7 receptors), which are expressed in the lung. Considering the protective importance of 5-HT receptor antagonists against development of pulmonary fibrosis, we evaluated whether 5-HT7 receptor antagonist (SB-269970) modulates lung inflammatory and fibrogenic processes in comparison with 5-HT2A/B receptor antagonist (terguride), in bleomycin (BLM)-induced idiopathic pulmonary fibrosis (IPF) model. IPF model induced by a single dose of intra-tracheal BLM instillation (5mg/kg), and rats were treated with intraperitoneal injection of SB-269970 (1mg/kg day) or terguride (1.2mg/kg/d). The experiment was carried out on two separate sets of rats that were killed at day 7th and day 21st to evaluate the endpoint of the IPF inflammatory and fibrogenic phases, respectively. During the inflammatory phase 5-HT2A/B and 5-HT7 receptor antagonists attenuated the BLM-induced increase in the lung fluid content, the inflammatory cytokines levels and oxidative stress burden. In the fibrogenic phase, both SB-269970 and terguride reduced the serotonin concentrations in lung homogenates and significantly protected against IPF fibrogenic phase by attenuating collagen deposition and mRNA expression of both transforming growth factor-ß1 (TGF- ß1), and procollagen type Ӏ (PINP). 5-hydroxytryptamine 5-HT7 receptor antagonist showed more benefits than 5-HT2A/B receptor antagonist on the deleterious effects accompanied BLM instillation. The present study showed involvement of 5-HT7 receptor in the pathophysiology of BLM-induced IPF in rats and identified it as a potential therapeutic target in lung fibrotic disorders.

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

RATIONALE: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. OBJECTIVE: To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. METHODS AND RESULTS: Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. CONCLUSIONS: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

Differential protein kinase C isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling.

RATIONALE: Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (I(KAChC)) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. OBJECTIVE: To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced I(KAChC) activation. METHODS AND RESULTS: Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I(KAChC) single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated I(KAChC), mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K+ current (I(KACh)) in inside-out patches. TP and ATR downregulated PKCα (by 33% and 37%, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 µmol/L) or inhibiting the Ca2+-dependent protease calpain with PD150606 (20 µmol/L) prevented PKCα downregulation and TP enhancement of I(KAChC). PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced I(KAChC) activation, whereas cPKC inhibition enhanced I(KAChC) activity in 1-Hz cells. CONCLUSIONS: PKC isoforms differentially modulate I(KACh), with conventional Ca(2+)-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca2+/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I(KAChC). These findings provide novel insights into mechanisms underlying I(KAChC) dysregulation in AF.

Voltage-clamp-based methods for the detection of constitutively active acetylcholine-gated I(K,ACh) channels in the diseased heart.

Vagal nerve stimulation can promote atrial fibrillation (AF) that requires activation of the acetylcholine (ACh)-gated potassium current I(K,ACh). In chronic AF (cAF), I(K,ACh) shows strong activity despite the absence of ACh or analogous pharmacological stimulation. This receptor-independent, constitutive I(K,ACh) activity is suggested to represent an atrial-selective anti-AF therapeutic target, but the underlying molecular mechanisms are unknown. This chapter provides an overview of the voltage-clamp techniques that can be used to study constitutive I(K,ACh) activity in atrial myocytes and summarizes briefly the current knowledge about the potential underlying mechanism(s) of constitutive I(K,ACh) activity in diseased heart.

A difference in inward rectification and polyamine block and permeation between the Kir2.1 and Kir3.1/Kir3.4 K+ channels.

Inward rectification is caused by voltage-dependent block of the channel pore by intracellular Mg2+ and polyamines such as spermine. In the present study, we compared inward rectification in the Kir3.1/Kir3.4 channel, which underlies the cardiac current I(K,ACh), and the Kir2.1 channel, which underlies the cardiac current I(K,1). Sustained outward current at potentials positive to the K+ reversal potential was observed through Kir3.1/Kir3.4, but not Kir2.1, demonstrating that Kir3.1/Kir3.4 exhibits weaker inward rectification than Kir2.1. We show that Kir3.1/Kir3.4 is more sensitive to extracellular spermine block than Kir2.1, and that intracellular and extracellular polyamines can permeate Kir3.1/Kir3.4, but not Kir2.1, to a limited extent. We describe a simple kinetic model in which polyamines act as permeant blockers of Kir3.1/Kir3.4, but as relatively impermeant blockers of Kir2.1. The model shows the difference in sensitivity to extracellular spermine block, as well as the difference in the extent of inward rectification between the two channels. This suggests that Kir3.1/Kir3.4 exhibits weaker inward rectification than Kir2.1 because of the difference in the balance of polyamine block and permeation of the two channels.

The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel.

The Kir3.1/Kir3.4 channel is activated by Gbetagamma subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel.

Molecular basis of ion selectivity, block, and rectification of the inward rectifier Kir3.1/Kir3.4 K(+) channel.

The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.