Effects of streptozotocin-induced diabetes on Kv channels in rat small coronary smooth muscle cells

Diabetes impairs endothelium dependent vasodilation, but the mechanism of endothelium independent dilation is not well understood. In the present study, we examined the effect of streptozotocin (STZ)-induced diabetes on the vasomotor of small coronary artery and the activity of voltage-dependent K+ channel of vascular smooth muscle cells in STZ rats [corrected] using the videomicroscopy and patch clamp method. STZ-induced diabetes appeared to [corrected] reduce the vasodilation induced by beta-adrenoceptor agonist, isoproterenol (10(-9)-10(-5) mol/l), and adenylyl cyclase activator forskolin (10(-9)-10(-5) mol/l) respectively (isoproterenol: 44.2 +/- 6.7% vs. 82.5 +/- 4.8%, and forskolin: 54.4 +/- 4.5% vs. 94.3 +/- 2.4%). 4-AP, a Kv channel blocker of VSMC, further decreased dilation to isoproterenol (44.2 +/- 6.7% vs. 10.2 +/- 3.5%) and forskolin (54.4 +/- 4.5% vs. 13.8 +/- 11.0%) significantly. Whole cell K+ current recording demonstrated that STZ-induced diabetes decreased isoproterenol and forskolin-induced K+ current (ISO: 55.6 +/- 7.8 pA/pF vs. 28.4 +/- 3.4 pA/pF, forskolin: 61.3 +/- 9.8 pA/pF vs. 32.4 +/- 3.4 pA/pF). 4-AP further reduced the decreased K+ current (ISO: 28.4 +/- 3.4 pA/pF vs. 14.3 +/- 2.1 pA/pF, forskolin: 32.4 +/- 3.4 pA/pF vs. 14.8 +/- 2.9 pA/pF). These results indicated that STZ-induced diabetes impaired cAMP mediated dilation of small coronary artery and suppressed the Kv channel activity of vascular smooth muscle cells. Kv channel of VSMC was shown to play a determinate role reducing dilation of small coronary artery in STZ rats.     

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.     

Does ghrelin explain accelerated gastric emptying in the early stages of diabetes mellitus?

During the early stages of diabetes, gastric emptying is often accelerated, rather than delayed. The mechanism of accelerated gastric emptying in diabetes has not been fully studied. A recent study showed that plasma ghrelin levels were elevated in diabetes. As postprandial antropyloric coordination plays an important role in mediating solid gastric emptying, we hypothesize that the elevated plasma ghrelin levels increase postprandial antropyloric coordination to accelerate emptying in the early stages of diabetes. To test this hypothesis, rats were made diabetic by streptozotocin (STZ; 50 mg/kg) injection, and, 2 wk later, pre- and postprandial plasma ghrelin levels, antropyloric coordination, and solid gastric emptying were determined. In control rats, plasma ghrelin levels were immediately reduced after feeding. In contrast, plasma ghrelin levels remained within the fasted levels in STZ rats after feeding. In STZ rats, gastric emptying was significantly accelerated (77.4 +/- 3.2%, n = 6), compared with that of control rats (58.8 +/- 2.5%, n = 6, P < 0.05). Treatments with anti-ghrelin antibodies attenuated accelerated gastric emptying in STZ rats (50.1 +/- 3.5%, n = 6, P < 0.05), while having little effect in vehicle control rats. The incidence of postprandial antropyloric coordination was significantly increased in STZ rats, compared with that of control rats (P < 0.05). Treatments with anti-ghrelin antibodies suppressed this enhanced antropyloric coordination in STZ rats. Our study suggests that elevated endogenous ghrelin enhances antropyloric coordination, which accelerates gastric emptying in the early stages of diabetes.     

Upregulation of small GTPase RhoA in the basilar artery from diabetic (mellitus) rats

The goal of this study was to determine whether RhoA, a small GTPase, might be involved in the development of cerebral pathogenesis in diabetes. Male SD rats (n = 120) were divided into six groups: diabetic for 2, 4, 8 weeks, and an age-matched control group. Diabetes was induced by intravenous injection of streptozotocin (50 mg/kg). RhoA mRNA expression in basilar artery was measured by competitive RT-PCR. RhoA mRNA level was significantly increased in 4 weeks (184.1 +/- 28.5%, n = 7) and 8 weeks (218.7 +/- 24.5%, n = 7) after STZ injection compared to the age matched control basilar arteries (P < 0.05). Western blot was used to measure the membrane binding RhoA level to represent the activity of RhoA. We found that RhoA activity was strikingly increased in the diabetic basilar artery (n = 10 in each groups) compared to control basilar artery after STZ injection. Our data demonstrated that there was an upregulation of RhoA in the basilar artery of STZ induced diabetic rats, suggesting that RhoA might be involved in the cerebral vascular pathogenesis during diabetes mellitus.     

Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat

The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent (86)Rb/K uptake was reduced to 60.0 +/- 5.5% of the control value (P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (alpha(1)) and ouabain-sensitive (alpha(2) and alpha(3)) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10(-3) M) or a low (10(-5) M) ouabain concentration during (86)Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated (86)Rb/K uptake (26.0 +/- 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of alpha(1)- and alpha(2)-catalytic isoforms (alpha(1) = 71.3 +/- 9.8% of control values, P < 0.05; alpha(2) = 44.5 +/- 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 +/- 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent (86)Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.     

Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes

In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P 相似文献   

Diabetes-related changes in cAMP-dependent protein kinase activity and decrease in relaxation response in rat mesenteric artery

Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283-H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6,O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-alpha were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-beta were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an imbalance between PKA catalytic and regulatory subunit expressions.     

Adenosine triphosphatase activity of streptozotocin-induced diabetic rat brain microsomes. Effect of vitamin E

Hyperglycemia causes protein glycosylation, oxidation and alterations in enzyme activities, which are the underlying causes of diabetic complications. This study was undertaken to test the role of vitamin E treatment on Ca2+-ATPase activity, protein glycosylation and lipid peroxidation in the brain of streptozotocin (STZ)-induced diabetic rats. Male rats weighing about 250-300 g were rendered diabetic by a single STZ injection of 50 mg/kg via the tail vein. Both the diabetic and non-diabetic rats were fed a vitamin E supplemented diet (500 IU/kg/day). Ca2+-ATPase activity was significantly reduced at week 10 of diabetes compared to the control group (p < 0.05), with 0.225+/-0.021 U/I (mean +/- S.E.M.) in the control group and 0.072 +/- 0.008 U/l (mean +/- S.E.M.) in the diabetic group. Vitamin E treatment prevented the enzyme activity from decreasing. The activities observed were 0.226 +/- 0.020 U/l and 0.172 +/- 0.011 U/I (mean +/- S.E.M.) in the vitamin E-treated control and diabetic group, respectively. STZ-induced diabetes resulted in an increased protein glycosylation and lipid peroxidation. Vitamin E treatment led to a significant inhibition in blood glucose, protein glycosylation and lipid peroxidation, which in turn prevented abnormal activity of the enzyme in the brain. This study indicates that vitamin E supplementation may reduce complications of diabetes in the brain.     

Protective effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation and antioxidant enzymes in diabetic rat liver

The aim of this study was to examine the effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation (LPO) and the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the liver of streptozotocin (STZ)-induced diabetic rats. Twenty-seven rats were randomly divided into three groups: group I, control non-diabetic rats (n = 9); group II, STZ-induced, untreated diabetic rats (n = 8); group III, STZ-induced, CAPE-treated diabetic rats (n = 10), which were intraperitoneally injected with CAPE (10 microM kg(-1) day(-1)) after 3 days followed by STZ treatment. The liver was excised after 8 weeks of CAPE treatment, the levels of malondialdehyde (MDA) and the activities of SOD, CAT, and GSH-Px in the hepatic tissues of all groups were analyzed. In the untreated diabetic rats, MDA markedly increased in the hepatic tissue compared with the control rats (p < 0.0001). However, MDA levels were reduced to the control level by CAPE. The activities of SOD, CAT, and GSH-Px in the untreated diabetic group were higher than that in the control group (p < 0.0001). The activities of SOD and GSH-Px in the CAPE-treated diabetic group were higher than that in the control group (respectively, p < 0.0001, p < 0.035). There were no significant differences in the activity of CAT between the rats of CAPE-treated diabetic and control groups. Rats in the CAPE-treated diabetic group had reduced activities of SOD and CAT in comparison with the rats of untreated diabetic group (p < 0.0001). There were no significant differences in the activity of GSH-Px between the rats of untreated diabetic and CAPE-treated groups. It is likely that STZ-induced diabetes caused liver damage. In addition, LPO may be one of the molecular mechanisms involved in STZ-induced diabetic damage. CAPE can reduce LPO caused by STZ-induced diabetes.     

骨形态发生蛋白-7及抑制性Smads在糖尿病肾病发生发展中的表达变化

本文采用免疫组化、Western blot及荧光实时定量PCR方法,动态观察链脲佐菌素（streptozocin,STZ）诱导的大鼠糖尿病肾病（diabetic nephropathy,DN）发生早期肾脏骨形态发生蛋白-7（bone morphogenetic protein-7,BMP-7）、Smad6、Smad7蛋白及mRNA表达。结果显示,在正常及DN大鼠肾小管均有BMP-7、Smad6、Smad7蛋白表达,以胞浆表达为主。DN大鼠BMP-7、Smad6蛋白表达较正常大鼠明显增多（P〈0．05）,且BMP-7的mRNA表达呈先增加后降低的状态;而Smad7蛋白和mRNA的表达均呈先增加后降低的状态。转化生长因子-β1（transforming growth factor-β1,TGF-β1）及Ⅰ型胶原（collagen typeⅠ,COL-Ⅰ）mRNA在DN大鼠肾脏表达较正常大鼠明显增多（P〈0．05）,且随着糖尿病进展有逐渐增加的趋势。结果提示,作为TGF-β超家族信号分子的一员,BMP-7信号及抑制性Smad通路在DN肾纤维化发生早期可能起重要的反馈性抑制作用。     

The effects of insulin-like growth factor binding protein-3 (IGFBP-3) on T47D breast cancer cells enriched for IGFBP-3 binding sites

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca2+) and magnesium (Mg2+) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg(-1), I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg(-1) urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca2+ and Mg2+ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 +/- 2.42 mg dl(-1) (n = 44) and >500 mg dl(-1) (n = 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 +/- 0.05 ul min(-1) (n = 10) and 1.28 +/- 0.16 ul min(-1) (n = 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca2+]i in control and diabetic fura-2-loaded acinar cells was 109.40 +/- 15.41 nM (n = 15) and 130.62 +/- 17.66 nM (n = 8), respectively. CCK-8 (10(-8)M) induced a peak response of 436.55 +/- 36.54 nM (n = 15) and 409.31 +/- 34.64 nM (n = 8) in control and diabetic cells, respectively. Basal [Mg2+]i in control and diabetic magfura-2-loaded acinar cells was 0.96 +/- 0.06 nM (n = 18) and 0.86 +/- 0.04 nM (n = 10). In the presence of CCK-8 (10(-8)) [Mg2+]i in control and diabetic cells was 0.80 +/- 0.05 nM (n = 18) and 0.60 +/- 0.02 nM (n = 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca2+ and Mg2+ homeostasis.     

Kv1.3 channels in postganglionic sympathetic neurons: expression, function, and modulation

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n >or= 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function.     

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro . It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kα (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1β and TNF-α mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1β and TNF-α expression and ROS production probably by regulating the intracellular potassium concentration.     

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Pulmonary vascular K+ channel expression and vasoreactivity in a model of congenital heart disease

K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K Ca ) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K Ca channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K Ca channel expression is increased and TASK is decreased.     

External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.     

Impaired responsiveness of renal sensory nerves in streptozotocin-treated rats and obese Zucker diabetic fatty rats: role of angiotensin

Increasing afferent renal nerve activity decreases efferent renal nerve activity and increases urinary sodium excretion. Activation of renal pelvic mechanosensory nerves is impaired in streptozotocin (STZ)-treated rats (model of type 1 diabetes). Decreased activation of renal sensory nerves would lead to increased efferent renal nerve activity, sodium retention, and hypertension. We examined whether the reduced activation of renal sensory nerves in STZ rats was due to increased renal angiotensin activity and whether activation of the renal sensory nerves was impaired in obese Zucker diabetic fatty (ZDF) rats (model of type 2 diabetes). In an isolated renal pelvic wall preparation from rats treated with STZ for 2 wk, PGE2 failed to increase the release of substance P, from 5 +/- 1 to 6 +/- 1 pg/min. In pelvises from sham STZ rats, PGE2 increased substance P release from 6 +/- 1 to 13 +/- 2 pg/min. Adding losartan to the incubation bath increased PGE2-mediated release of substance P in STZ rats, from 5 +/- 1 to 10 +/- 2 pg/min, but had no effect in sham STZ rats. In pelvises from obese ZDF rats (22-46 wk old), PGE2 increased substance P release from 12.0 +/- 1.2 to 18.3 +/- 1.2 pg/min, which was less than that from lean ZDF rats (10.3 +/- 1.6 to 22.5 +/- 2.4 pg/min). Losartan had no effect on the PGE2-mediated substance P release in obese or lean ZDF rats. We conclude that the mechanisms involved in the decreased responsiveness of the renal sensory nerves in STZ rats involve activation of the renin angiotensin system in STZ but not in obese ZDF rats.