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Y Shimoni

Publications and source records attributed to Y Shimoni.

At least 19 recordsLinked to original sources
PubMed2006

Propagation of the cardiac impulse in the diabetic rat heart: reduced conduction reserve.

Diabetes mellitus is a growing epidemic with severe cardiovascular complications. Although much is known about mechanical and electrical cardiac dysfunction in diabetes, few studies have investigated propagation of the electrical signal in the diabetic heart and the associated changes in intercellular gap junctions. This study was designed to investigate these issues, using hearts from control and diabetic rats. Diabetic conditions were induced by streptozotocin (STZ), given i.v. 7-14 days before experiments. Optical mapping with the voltage-sensitive dye di-4-ANEPPS, using hearts perfused on a Langendorff apparatus, showed little change in baseline conduction velocity in diabetic hearts, reflecting the large reserve of function. However, both the gap junction uncoupler heptanol (0.5-1 mM) and elevated potassium (9 mM, to reduce cell excitability) produced a significantly greater slowing of impulse propagation in diabetic hearts than in controls. The maximal action potential upstroke velocity (an index of the sodium current) and resting potential was similar in single ventricular myocytes from control and diabetic rats, suggesting similar electrical excitability. Immunoblotting of connexin 43 (Cx43), a major gap junction component, showed no change in total expression. However, immunofluorescence labelling of Cx43 showed a significant redistribution, apparent as enhanced Cx43 lateralization. This was quantified and found to be significantly larger than in control myocytes. Labelling of two other gap junction proteins, N-cadherin and beta-catenin, showed a (partial) loss of co-localization with Cx43, indicating that enhancement of lateralized Cx43 is associated with non-functional gap junctions. In conclusion, conduction reserve is smaller in the diabetic heart, priming it for impaired conduction upon further challenges. This can desynchronize contraction and contribute to arrhythmogenesis.

Animals↗
PubMed2006

Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin.

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dt(max)) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (I(Na)) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of I(Na). All of these treatments rescued I(Na) to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of I(Na). To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.

Action Potentials↗
PubMed2005

Modulation of potassium currents by angiotensin and oxidative stress in cardiac cells from the diabetic rat.

Diabetes induces oxidative stress and leads to attenuation of cardiac K+ currents. We investigated the role of superoxide ions and angiotensin II (ANG II) in generating and linking oxidative stress to the modulation of K+ currents under diabetic conditions. K+ currents were measured using patch-clamp methods in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats. Superoxide ion levels, indicating oxidative stress, were measured by fluorescent labelling with dihydroethidium (DHE). ANG II content was measured using enzyme-linked immunosorbent asssay (ELISA). The results showed DHE fluorescence to be significantly higher in cells from diabetic males, compared to controls. Relief of stress by the NADPH oxidase inhibitor apocynin or by superoxide dismutase (SOD) but not by catalase reversed the attenuation of K+ currents and reduced DHE fluorescence. In cells from diabetic females, neither apocynin nor SOD augmented K+ currents, ANG II was not elevated and DHE fluorescence was significantly weaker than in cells from males. Reduced glutathione (GSH) also augmented K+ currents in cells from diabetic males but not females. In ovariectomized diabetic females K+ currents were augmented by GSH and apocynin. Current augmentation and the attenuation of DHE fluorescence by apocynin were significantly blunted by excess ANG II (300 nm). Diabetic male rats pretreated with the angiotensin-converting enzyme (ACE) inhibitor quinapril were hyperglycaemic, but their cellular ANG II levels and DHE fluorescence were significantly decreased. In cells from these rats, K+ currents were insensitive to apocynin. In conclusion, diabetes-related oxidative stress attenuates K+ currents through ANG II-generated increased superoxide ion levels. When ANG II levels are lower, as in diabetic females or following ACE inhibition in males, oxidative stress is reduced, with blunted alterations in K+ currents.

Angiotensin II↗
PubMed2005

Solid tissues can be manipulated ex vivo and used as vehicles for gene therapy.

BACKGROUND: Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. METHODS: MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene beta-galactosidase. RESULTS: All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (> 15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. CONCLUSIONS: The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance.

Animals↗
PubMed2001

Inhibition of the formation or action of angiotensin II reverses attenuated K+ currents in type 1 and type 2 diabetes.

1. Transient and sustained calcium-independent outward K(+) currents (I(t) and I(SS)) as well as action potentials were recorded in cardiac ventricular myocytes isolated from two models of diabetes mellitus. 2. Rats injected (I.V.) with streptozotocin (STZ, 100 mg kg(-1)) 6-10 days before cell isolation developed insulin-dependent (type 1) diabetes. I(t) and I(SS) were attenuated and the action potential prolonged. Incubation of myocytes (6-9 h) with the angiotensin II (ATII) receptor blockers saralasin or valsartan (1 microM) significantly augmented these currents. Inclusion of valsartan (1 g l(-1)) in the drinking water for 5-10 days prior to and following STZ injection partially prevented current attenuation. 3. Incubation of myocytes from STZ-treated rats (6-9 h) with 1 microM quinapril, an angiotensin-converting enzyme (ACE) inhibitor, significantly augmented I(t) and I(SS) and shortened the ventricular action potential. I(t) augmentation was not due to changes in steady-state inactivation or in recovery from inactivation. No acute effects of quinapril were observed. 4. The effects of quinapril and valsartan were abolished by 2 microM cycloheximide. 5. Myocytes were isolated from the db/db mouse, a leptin receptor mutant that develops symptoms of non-insulin-dependent (type 2) diabetes. K+ currents in these cells were also attenuated, and the action potentials prolonged. Incubation of these cells (> 6 h) with valsartan (1 microM) significantly enhanced the transient and sustained outward currents. 6. These results confirm recent suggestions that cardiac myocytes contain a renin-angiotensin system, which is activated in diabetes. It is proposed that chronic release of ATII leads to changes in ionic currents and action potentials, which can be reversed by blocking the formation or action of ATII. This may underlie the proven benefits of ATII receptor blockade or ACE inhibition in diabetes, by providing protection against cardiac arrhythmias.

Action Potentials↗
PubMed2001

Transmural differences in rat ventricular protein kinase C epsilon correlate with its functional regulation of a transient cardiac K+ current.

The effects of PKC activation on a transient (It) and a sustained (Iss) cardiac K+ current and the subcellular distribution of the epsilon isoform of PKC (PKC(epsilon)) were compared in epicardial and endocardial regions of the rat ventricle. Activation of PKC(epsilon) with a diacylglycerol analogue (di-octanoyl-glycerol (DiC8), 20 (mu)M) leads to differential effects in epicardial and endocardial cells. In epicardial cells (n = 20) It and Iss are attenuated by 17.7 +/- 2.1 % and 11.9 +/- 3.1 %, respectively (means +/- S.E.M.). In endocardial cells It attenuation was significantly smaller (4.6 +/- 1.6 %, n = 14, P < 0.0005). Iss attenuation was similar to that in epicardial cells (10.5 +/- 3.8 %). PKC[epsilon] expression was measured by Western blotting. Calculated endocardial/epicardial ratios showed no regional differences in total protein extracts (1.04 +/- 0.11, mean +/- S.E.M, n = 4), but PKC[epsilon] distribution in the cytosolic fraction showed a marked difference, with significantly (P < 0.05) higher levels in endocardial extracts. The cytosolic endocardial/epicardial PKC[epsilon] ratio was 2.64 +/- 0.24 (n = 4), indicating a reduced amount of PKC[epsilon] in the membrane fraction of the endocardium. This could account for the reduced effect of DiC8 on It in endocardial myocytes. Under both hypothyroid and streptozotocin-induced diabetic conditions the difference in endocardial and epicardial cytosolic PKC[epsilon] levels was absent (ratios of 0.86 +/- 0.21 (n = 4) and 1.09 +/- 0.16 (n = 3), respectively; means +/- S.E.M.). Ratios in the total protein extracts were not significantly different from those in control conditions. The results show transmural differences in the functional effects of PKC(epsilon) activation on a cardiac K+ current, and in the subcellular distribution of PKC(epsilon). These differences are absent in diabetic and hypothyroid conditions.

Animals↗
PubMed2001

Type 1 diabetes leads to cytoskeleton changes that are reflected in insulin action on rat cardiac K(+) currents.

A sustained K(+) current (I(ss)) is attenuated in ventricular cells from streptozotocin (STZ)-induced diabetic rats. The in vitro addition of insulin to isolated cells augments I(ss) in a process that is blocked by disrupting either actin microfilaments (with cytochalasin D) or microtubules (with colchicine). When these agents are added at progressively later times, the effect of insulin becomes evident in a time-dependent manner. I(ss) is also augmented by insulin in control cells in a cytoskeleton-dependent manner. However, in contrast to diabetic cells, cytoskeleton-dependent augmentation of I(ss) by insulin occurs at a considerably faster rate in control cells. Immunofluorescent labeling shows a reduced density of beta-tubulin in diabetic cells, particularly in perinuclear regions. In vitro insulin replacement or in vivo insulin injections given to STZ-treated rats enhances beta-tubulin density. These results suggest an impairment of cytoskeleton function and structure under insulin-deficient conditions, which may have implications for cardiac function.

Animals↗
PubMed2000

Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae.

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.

Adenosine Triphosphatases↗
PubMed2000

Insulin resistance and the modulation of rat cardiac K(+) currents.

K(+) currents were measured using a whole cell voltage-clamp method in enzymatically isolated rat ventricular myocytes obtained from two hyperinsulinemic, insulin-resistant models. Fructose-fed rats as well as genetically obese rats, both of which are resistant to the metabolic effects of insulin, were used. The normal augmentation of a calcium-independent sustained K(+) current was reduced or abolished in insulin-resistant states. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadyl sulfate (3-4 wk treatment or after 5-6 h in vitro) enhanced the sustained K(+) current. The in vitro effect of vanadyl was blocked by cycloheximide. Insulin resistance of the K(+) current was not reversed by vanadyl sulfate. The results show that insulin resistance is expressed in terms of insulin actions on ion channels, in addition to its actions on metabolism. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadate compounds, which mimic the effects of insulin on metabolism, also mimic the augmenting effects of insulin on a cardiac K(+) current in a manner suggesting synthesis of new channels.

Animals↗
PubMed1999

Protein kinase C regulation of K+ currents in rat ventricular myocytes and its modification by hormonal status.

1. The effects of protein kinase C (PKC) activation on cardiac K+ currents were studied in rat ventricular myocytes, using whole-cell voltage clamp methods. Control rats were compared to hypothyroid or diabetic rats, in which PKC expression and activity were enhanced. 2. In control myocytes, two calcium-independent outward K+ currents, the transient It and the sustained Iss, were attenuated by 18.9 +/- 2.0 and 16.8 +/- 3.5 %, respectively (mean +/- s.e.m.), following addition of a synthetic analogue of diacylglycerol, DiC8 (20 microM). In myocytes from hypothyroid or diabetic rats, It and Iss were not affected by DiC8. 3. The effects of DiC8 were restored in myocytes from thyroidectomized rats by injection of physiological doses of tri-iodothyronine (T3; 10 microg kg-1 for 6-8 days). Incubating cells from diabetic rats with 100 nM insulin for 5-9 h also restored the ability of DiC8 to attenuate It and Iss. 4. The attenuation of K+ currents by DiC8 in control cells was absent in the presence of a peptide known to inhibit the translocation of the isoform PKCepsilon (EAVSKPLT, 24 microM introduced through the recording pipette). A scrambled peptide (LSETKPAV) was without effect. 5. Under hypothyroid conditions the inhibitory peptide restored the effects of DiC8 on It and Iss. These currents were attenuated by 11.9 +/- 1. 5 and 9.8 +/- 1.5 %, respectively, which was significantly (P < 0. 001) more than without the peptide or with the scrambled peptide. 6. These results show that the PKC-mediated suppression of cardiac K+ currents is normally mediated by PKCepsilon translocation. This effect is absent under hypothyroid and diabetic conditions, presumably due to prior PKC activation and translocation. A PKCepsilon translocation inhibitor restores the ability of DiC8 to attenuate K+ currents under hypothyroid conditions. This presumably reflects a (partial) reversal of a chronic translocation and a shift in the balance between PKC and its anchoring proteins.

Animals↗
PubMed1999

Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton.

1. The effect of insulin on K+ currents was studied with enzymatically dispersed ventricular myocytes from insulin-deficient (type I) diabetic rats. Diabetic conditions were induced by a single intravenous injection of streptozotocin (100 mg kg-1) given 8-13 days before the experiments. Measurements of plasma glucose and insulin levels confirmed the diabetic status of the animals. 2. A Ca2+-independent transient outward K+ current, It, and a slowly inactivating, quasi-steady-state current, Iss, which are depressed in diabetic myocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5-6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on It and Iss. The stimulatory effect of insulin on these K+ currents was prevented by 2 microM cycloheximide, which in itself had no direct effect on these currents. 3. Disruption of the actin microfilament network with 1 microM cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both It and Iss. Since CD was added 1 h after insulin, inhibitory effects on insulin signalling were ruled out. Adding CD (1 microM) 5-9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Incubating control cells for 6-10 h with 1 microM CD had no effect on any of the currents measured. 4. Stabilization of the actin network by pre-exposure to 2.5 microM phalloidin restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5. The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule-disrupting agent colchicine (5 microM). 6. These results suggest that the insulin-mediated augmentation of K+ currents in diabetic myocytes requires protein synthesis, possibly of K+ channels, as well as an intact cytoskeleton. The possibility that newly formed channels translocate to the plasma membrane in a process dependent on different elements of the cytoskeleton is discussed.

Actins↗
PubMed1998

Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node.

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Hypothyroidism decreases the ATP sensitivity of KATP channels from rat heart.

The effects of thyroid status on the properties of ATP-sensitive potassium channels were investigated. Single-channel recordings were made using excised inside-out membrane patches from enzymatically dissociated ventricular myocytes from hearts of control and thyroidectomized rats and each group was studied with and without administration of thyroid hormone. In patches excised from hypothyroid myocytes the IC50 for ATP inhibition of KATP channels was 110 micro m. This value was 3-fold higher than the IC50 in control myocytes (43 micro m). Treatment of hypothyroid rats to restore physiological levels of thyroid hormone (tri-iodothyronine, T3), resulted in a return to normal ATP-sensitivity (IC50 = 46 micro M). In patches from animals rendered hyperthyroid, the IC50 for ATP was 50 micro M and this value was not significantly different from the control. There was no difference in the cooperativity of ATP-binding (Hill coefficient, nH) among control (nH = 2.2), hypothyroid (nH = 2.1), T3-treated (nH = 2.0) and hyperthyroid groups (nH = 2.4). The unitary conductance was unchanged and there was no apparent change in intraburst kinetics between examples of single KATP channels from control and hypothyroid rats. Action potentials recorded in myocytes from hypothyroid rats were significantly shortened by 50 micro M levcromakalim, a KATP channel opener (P < 0.001) but unchanged in control myocytes.We conclude that hypothyroidism significantly decreased the ATP-sensitivity of KATP channels, whereas the induction of hyperthyroid conditions did not alter the ATP-sensitivity of these channels. Thus, hypothyroidism is likely to have important physiological consequences under circumstances in which KATP channels are activated, such as during ischemia.

Action Potentials↗
PubMed1998

Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin.

1. Several K+ currents were measured and compared in enzymatically dispersed ventricular myocytes from control and diabetic rats. 2. Diabetic conditions were established either with a single intravenous injection of streptozotocin (STZ, 100 mg kg-1; 6-14 days duration) or by feeding with a fructose-enriched diet for 4-10 weeks. Both groups became hyperglycaemic, with the former having decreased and the latter having elevated levels of plasma insulin. These conditions therefore mimic type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus, respectively. 3. As reported previously, a Ca(2+)-independent transient outward K+ current, I(t), was attenuated in the type I model. This was not observed in the type II model. The two models differed greatly in the changes observed in a quasi-steady-state K+ current denoted Iss. In the STZ model Iss was substantially attenuated, whereas in the fructose-fed model it was augmented. In both models, the background inwardly rectifying current, IK1, was unchanged. Concomitantly, there was a substantial prolongation of the action potential in the STZ model but not in the fructose-fed model. 4. Incubation of control myocytes with insulin (100 nM) for 5-9 h caused a significant augmentation of Iss, with no effect on I(t) or on IK1. Incubation of myocytes from STZ-diabetic rats with insulin reversed the attenuation of I(t), but not of Iss. 5. The effect of insulin was not blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. However, inhibition of the mitogen-activated protein kinase pathway with PD98059 prevented restoration of I(t). Insulin action on I(t) may therefore involve changes in transcription or expression of channel proteins, rather than changes in cellular metabolism.

Androstadienes↗
PubMed1998

Altered ATP sensitivity of ATP-dependent K+ channels in diabetic rat hearts.

The effects of streptozotocin-induced diabetes (5-7 days or 7 wk) on cardiac ATP-sensitive potassium channels (KATP channels) were investigated with the use of single-channel and action potential recordings from dissociated ventricular myocytes isolated from control and diabetic rat hearts. In inside-out patches from diabetic myocytes (5-7 days), the IC50 for ATP inhibition was 82 +/- 7.2 microM (mean +/- SE, n = 8), twice that in controls (43 +/- 3.6 microM, n = 12). For 7-wk diabetic rats, the IC50 was 75 +/- 2.3 microM (n = 6). Increasing internal ADP concentration attenuated ATP-induced inhibition in both controls and diabetics. On reducing the internal pH from 7.4 to 6.8, both control and diabetic myocytes showed a 1.7-fold increase in the IC50 for ATP inhibition. No differences were observed in either intraburst kinetics or unitary conductance of single channels from control and diabetic myocytes. In diabetic myocytes, action potential duration at 90% repolarization (APD90) was longer and more variable than in controls and was significantly shortened by application of the KATP channel opener cromakalim (50 microM). Cromakalim scarcely affected APD90 in controls. Computer simulation of the longer diabetic APD90 required a lower background conductance during the plateau phase in addition to small, measured changes in the delayed rectifier current, transient outward current, and ATP-sensitive K+ current (KATP current, IKATP). The simulations reproduced the enhanced sensitivity of the diabetic APD90 to changes in IKATP. These results have important implications for cardiac function in diabetics and their treatment by sulfonylureas.

Action Potentials↗
PubMed1997

A recombinant protein of two high molecular weight glutenins alters gluten polymer formation in transgenic wheat.

Wheat high molecular weight glutenin subunits (HMW-GS) are the most important determinants of its superiority for making leavened bread. Following synthesis, these proteins are sequestered into the endoplasmic reticulum and assemble into extremely large elastic polymers, linked by noncovalent and intermolecular disulfide bonds. To study the structural requirements for the assembly of HMW-GS, we have expressed in transgenic wheat a recombinant protein between two cognate x- and y-type subunits. In contrast to the natural polymerized x- and y-type HMW-GS, a significant amount of the recombinant subunit remained monomeric. Nonreducing SDS-polyacrylamide gel electrophoresis, coupled with limited proteolysis, showed that the monomeric form of the recombinant subunit contained an unusual intramolecular disulfide bond, linking an N-terminal cysteine to the single C-terminal cysteine residue. In addition, sucrose gradient analysis revealed that this intramolecular disulfide bond impeded the ability of the recombinant subunit to assemble into polymers. Despite of its altered assembly, a notable amount of the overexpressed recombinant subunit was also present in glutenin polymers. Moreover, its presence significantly altered the subunit composition of the polymer. Our results show that it is possible to modify gluten assembly and properties by expressing recombinant HMW-GS in transgenic wheat, and have a major implication for the improvement of wheat bread-making quality.

Amino Acid Sequence↗
PubMed1997

Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle.

1. The hypothesis that Kv4.2 and Kv4.3 are two of the essential K+ channel isoforms underlying the Ca2+-independent transient outward K+ current (It) in rat ventricle has been tested using a combination of electrophysiological measurements and antisense technology in both native myocytes and a stably transfected mammalian cell line, mouse Ltk- cells (L-cells). 2. The transient outward currents generated by Kv4.2 channels in L-cells exhibit rapid activation and inactivation properties similar to those produced by It in rat ventricular cells. The current-voltage relationships and the voltage dependence of steady-state inactivation are also very similar in these two preparations. However, the recovery from inactivation of Kv4.2 is much slower (time constant, 378 ms) than that of It in rat ventricular cells (58 ms). 3. The K+ current due to Kv4.2 can be blocked by millimolar concentrations of 4-aminopyridine in L-cells; a similar pharmacological response has been observed in rat ventricular myocytes. 4. Quinidine inhibits Kv4.2 in L-cells and It in rat ventricular cells in a similar fashion. In L-cells quinidine reduced the amplitude of Kv4.2 and accelerated its time course of inactivation, suggesting that quinidine may act as an open channel blocker of Kv4.2, as has been described for It in rat ventricle. 5. To provide further independent evidence that Kv4.2 and Kv4.3 channel isoforms contribute to It in rat ventricular cells, the effects of 20-mer antisense phosphorothioate oligodeoxynucleotides directed against Kv4.2 and Kv4.3 mRNAs were examined in ventricular myocytes isolated from 14- and 20-day-old rats, and in L-cells. In both preparations, Kv4.2 antisense pretreatment significantly reduced the transient outward K+ current (by approximately 55-60%). Similar reduction of It was produced by the Kv4.3 antisense oligonucleotide on the 14-day-old rat myocytes. 6. In 14-day rat ventricular cells, combination of Kv4.2 and Kv4.3 antisense oligonucleotides did not produce a significantly larger reduction of It than that observed after pretreatment with either antisense oligonucleotide alone. 7. L-cells stably transfected with Kv4.2 were treated with Kv4.3 antisense oligonucleotide to evaluate the possibility of cross-reactivity between Kv4.3 antisense and Kv4.2 mRNA. This antisense treatment produced no change in It, verifying the lack of cross-reactivity. 8. These biophysical and pharmacological results together with the antisense data show that Kv4.2 and Kv4.3 are essential components of the Ca2+-independent transient outward K+ current, It, in rat ventricular myocytes.

4-Aminopyridine↗