The importance of KATP channels in stimulus‐secretion coupling of β‐cells is well established, although they are not indispensable for the maintenance of glycaemic control. This review article ...depicts a new role for KATP channels by showing that genetic or pharmacological ablation of these channels protects β‐cells against oxidative stress. Increased production of oxidants is a crucial factor in the pathogenesis of type 2 diabetes mellitus (T2DM). T2DM develops when β‐cells can no longer compensate for the high demand of insulin resulting from excess fuel intake. Instead β‐cells start to secrete less insulin and β‐cell mass is diminished by apoptosis. Both, reduction of insulin secretion and β‐cell mass induced by oxidative stress, are prevented by deletion or inhibition of KATP channels. These findings may open up new insights into the early treatment of T2DM.
Aims/hypothesis
Evidence is accumulating that Ca
2+
-regulated K
+
(K
Ca
) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K
Ca
...channels for glucose homeostasis, beta cell function and viability.
Methods
Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca
2+
concentration (Ca
2+
c
) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay.
Results
BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of Ca
2+
c
. BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H
2
O
2
, 10–100 μmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H
2
O
2
-induced K
+
currents revealed that BK channels contribute to the hyperpolarising K
+
current activated under conditions of oxidative stress.
Conclusions/interpretation
Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus–secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.
The gastrooesophageal reflux disease (GERD) is a possible cause of chronic cough. The laparoscopic fundoplication is well established in the treatment of GERD. In a retrospective study, the ...effectivity of this operation on the GERD associated cough was examined and possible preoperative predictive factors concerning the post-surgical therapy effect were characterized.
85 patients after laparoscopic fundoplication due to GERD treated with proton pump inhibitors without (RS-H: n = 31) or with associated cough (RS+H: n = 54) were evaluated in a three-month follow-up by data analysis regarding an indication point score from typical symptoms as well as findings (gastroscopy, histology, 24-hour oesophagus pH-metry).
For the leading symptoms of heartburn and regurgitation a complete freedom from complaints was reached with 98.8 % of all patients postal-surgically. In the group RS+H 70.4 % of the patients were free of cough after 3 months, other 22.2 % with significant improvement and 7.4 % with unchanged irritant cough. Higher values of the typical reflux symptoms and a therapy resistance to proton pump inhibitors (PPI) were clearly seen in the RS-H patients. The RS+H patients showed less reflux complaints with lower PPI resistance, frequent allergies as well as significantly more often an acid or bitter taste and hoarseness. After further subdivision of the RS+H patients into the subgroups RS>H (mainly reflux, n = 31) and H>RS (mainly cough), the lowest values for heartburn, regurgitation and PPI resistance were found in subgroup H>RS. Diagnostics did not show any significiant differences between the groups although a trend could be seen towards fewer duodenogastric bile reflux, larger hiatus hernias and higher DeMeester scores in RS+H and H>RS. Also smokers, non-allergic asthmatics and polyallergic sufferers with cough profited from the intervention.
Patients with reflux-associated respiratory symptoms present an own entity with good PPI therapy response to heartburn, but not to cough. They should be considered more often for surgery. Since the cough symptoms in more than two-thirds of appropriately selected patients disappear in a short time after surgery, laparoscopic antireflux surgery should also be considered from pneumological aspects. There are no individual predictors for the success of antireflux surgery, only the sum of all relevant individual case history and clinical criteria, as they are combined in the used score, can provide a reliable indication for surgery.
SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with ...stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations (Ca(2+)(c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes. In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. Ca(2+)(c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and Ca(2+)(c) in cells from both SUR1(-/-) and SUR1(+/+) mice. The strong dependence of Vm and Ca(2+)(c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.
While the shoot apical meristem (SAM) is indirectly responsible for the initiation of all above-ground postembryonic organs, in most plants the vast majority of these organs are directly initiated by ...lateral meristems. In Arabidopsis thaliana, the lateral meristems include flower meristems (FMs), which form on the flanks of the SAM, and lateral shoot meristems (LSMs), which develop in leaf axils. While significant progress has been made on the molecular genetic basis of SAM initiation during embryo development, relatively little is known about the initiation of meristems at lateral positions. Here we have characterized the phenotypic consequences and genetic interactions of mutations in the REVOLUTA (REV) gene, with an emphasis on the role of REV in lateral meristem initiation. Our observations indicate that REV is required for initiation of both LSMs and FMs, and likely acts in the same pathway as, and upstream of, known meristem regulators. We identified the REV gene and found it encodes a predicted homeodomain/leucine zipper transcription factor that also contains a START sterol-lipid binding domain. REV is the same as the IFL gene. REV was expressed at the earliest stages of LSM and FM formation. Within the inflorescence shoot meristem, REV expression appeared to predict 3--5 incipient flower primordia on the flanks of the SAM, and REV expression at stage 1 and stage 2 matched that of WUS and STM, respectively. We propose that REV acts at lateral positions to activate the expression of known meristem regulators.
The plant life cycle alternates between a diploid sporophyte generation and
a haploid gametophyte generation. The angiosperm female gametophyte is critical
to the reproductive process. It is the ...structure within which egg cell
production and fertilization take place. In addition, the female gametophyte
plays a role in pollen tube guidance, the induction of seed development, and
the maternal control of seed development. Genetic analysis in Arabidopsis has
uncovered mutations that affect female gametophyte development and function.
Mutants defective in almost all stages of development have been identified, and
analysis of these mutants is beginning to reveal features of the female
gametophyte developmental program. Other mutations that affect female
gametophyte function have uncovered regulatory genes required for the induction
of endosperm development. From these studies, we are beginning to understand
the regulatory networks involved in female gametophyte development and
function. Further investigation of the female gametophyte will require
complementary approaches including expression-based approaches to obtain a
complete profile of the genes functioning within this critical structure.
Lateral organs produced by shoot apical and flower meristems exhibit a fundamental abaxial-adaxial asymmetry. We describe three members of the YABBY gene family, FILAMENTOUS FLOWER, YABBY2 and ...YABBY3, isolated on the basis of homology to CRABS CLAW. Each of these genes is expressed in a polar manner in all lateral organ primordia produced from the apical and flower meristems. The expression of these genes is precisely correlated with abaxial cell fate in mutants in which abaxial cell fates are found ectopically, reduced or eliminated. Ectopic expression of either FILAMENTOUS FLOWER or YABBY3 is sufficient to specify the development of ectopic abaxial tissues in lateral organs. Conversely, loss of polar expression of these two genes results in a loss of polar differentiation of tissues in lateral organs. Taken together, these observations indicate that members of this gene family are responsible for the specification of abaxial cell fate in lateral organs of Arabidopsis. Furthermore, ectopic expression studies suggest that ubiquitous abaxial cell fate and maintenance of a functional apical meristem are incompatible.
SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with ...stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial.
Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations (Ca(2+)(c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes.
In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. Ca(2+)(c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and Ca(2+)(c) in cells from both SUR1(-/-) and SUR1(+/+) mice.
The strong dependence of Vm and Ca(2+)(c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.
Aims/hypothesis In a previous study, we demonstrated that a creatine kinase (CK) modulates KATP channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more ...detail, we examined whether KATP channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. Methods Single channel activity was measured with the patch-clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. Results The ATP sensitivity of KATP channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in KATP channel regulation. The observation that the stimulatory effect of AMP on KATP channels was prevented by the AK inhibitor P ¹,P ⁵-di(adenosine-5')pentaphosphate (Ap₅A; 20 μmol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5'-(β,γ-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in KATP channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. Conclusions/interpretation We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell KATP channels. Thus, these enzymes determine the open probability of KATP channels and eventually the actual rate of insulin secretion.
This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this ...method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.
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