Macromolecular characterization of high ß-glucan oat lines.

Oat (Avena sativa) is a cereal grain rich in fibers, proteins, vitamins and minerals. Oats have been linked to several health benefits, such as lowering blood cholesterol levels, counteracting cardiovascular disease and regulating blood sugar levels. This study aimed to characterize two new oat lines with high ß-glucan content emanating from ethyl methyl sulphonate mutagenesis on the Lantmännen elite variety Belinda. Two of the mutated lines, and the mother variety Belinda, were profiled for ß-glucan, arabinoxylan, total dietary fiber and starch composition. In addition, total lipid and protein content, amino acid composition and ß-glucan molecular weights were analyzed. The high levels of ß-glucan resulted in a significant increase in total dietary fiber, but no correlation could be established between higher or lower levels of the assayed macromolecules, i.e., between arabinoxylan-, starch-, lipid- or protein levels in the mutated lines compared to the reference. The results indicate separate biosynthetic pathways for ß-glucans and other macromolecules and an independent regulation of the different polysaccharides studied. Therefore, ethyl methyl sulphonate mutagenesis can be used to increase levels of multiple macromolecules in the same line.

Pupil light reflex dynamics in Parkinson's disease.

Introduction: Visual disturbance is common symptom in Parkinson's disease (PD), and defective pupil light reflex (PLR) is an anticipated contributing factor that may be associated to the presence of autonomic dysfunction, which is a common non-motor feature of PD. Studies investigating the intercorrelation between PLR and dysautonomia in PD are limited. Methods: The aim of this study was to investigate differences of PLR parameters, measured by eye-tracker, between patients with PD, with and without signs of dysautonomia, and healthy controls (HC). In total, 43 HC and 50 patients with PD were recruited and PLR parameters were measured with Tobii Pro Spectrum, during a long (1,000 ms) and a short (100 ms) light stimulus. Presence of orthostatic hypotension (OH) was used as proxy marker of dysautonomia. Linear mixed-effects model and non-parametric comparative statistics were applied to investigate differences among groups. Results: Peak constriction velocity was slower in PD compared with HC, after adjustment for age and sex in the mixed model, and the difference was greater in the subgroup of PD with OH (unadjusted). Dilation amplitude and velocity were also gradually slower in HC vs. PD without OH vs. PD with OH (unadjusted for confounders). In the mixed model, age was significant predictor of dilation response. Discussion: Our results support previous observations on defective PLR in PD, evaluated with eye-tracker, and show a possible association with autonomic dysfunction. Further studies with more patients and rigorous evaluation of autonomic dysfunction are needed to validate these findings.

Reading Alterations in Parkinson's Disease Indicate Worse Cognitive Status.

Background: Reading difficulties are commonly reported in Parkinson's disease (PD). So far, only a few studies have assessed reading in PD, most of them confirming a different pattern in patients compared with healthy populations. Impaired oculomotor control is an early feature of PD. Cognitive deficits, on the other hand, may appear early, but they are most prominent at later stages. Although these two factors are thought to be responsible for the alterations in reading performance, it is unclear how each factor contributes to them. Objectives: To evaluate eye movements during reading in PD and healthy controls (HCs). Methods: Data from 42 HCs (36% men) and 48 patients with PD (67% men) at Hoehn and Yahr stages ≤3 were analyzed. PD participants were further divided into 2 groups based on their Montreal Cognitive Assessment (MoCA) score using a cutoff of ≥26. Eye movements were recorded with Tobii Pro Spectrum, a screen-based eye tracker with a sampling rate of 1200 Hz. Results: PD participants performed fewer fixations per second (P = 0.033), with a longer mean (P = 0.037) and standard deviation fixation duration (P = 0.033) than HC, and further analysis showed that only patients with a lower MoCA score performed worse than HCs. Reading parameters were weakly associated with MoCA scores, irrespective of age and education. Conclusion: Changes in the reading pattern of PD patients are probably attributed to cognitive rather than pure oculomotor alterations.

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties.

Background: The ATP-sensitive K+ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K+ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting ß-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K+ equilibrium potential around -80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics. Methods: We have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique. Results: Analyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current. Conclusions: Outward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels.

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Fixation Duration and Pupil Size as Diagnostic Tools in Parkinson's Disease.

BACKGROUND: Visual and oculomotor problems are very common in Parkinson's disease (PD) and by using eye-tracking such problems could be characterized in more detail. However, eye-tracking is not part of the routine clinical investigation of parkinsonism. OBJECTIVE: To evaluate gaze stability and pupil size in stable light conditions, as well as eye movements during sustained fixation in a population of PD patients and healthy controls (HC). METHODS: In total, 50 PD patients (66% males) with unilateral to mild-to-moderate disease (Hoehn & Yahr 1-3, Schwab and England 70-90%) and 43 HC (37% males) were included in the study. Eye movements were recorded with Tobii Pro Spectrum, a screen-based eye tracker with a sampling rate of 1200 Hz. Logistic regression analysis was applied to investigate the strength of association of eye-movement measures with diagnosis. RESULTS: Median pupil size (OR 0.811; 95% CI 0.666-0.987; p = 0.037) and longest fixation period (OR 0.798; 95% CI 0.691-0.921; p = 0.002), were the eye-movement parameters that were independently associated with diagnosis, after adjustment for sex (OR 4.35; 95% CI 1.516-12.483; p = 0.006) and visuospatial/executive score in Montreal Cognitive Assessment (OR 0.422; 95% CI 0.233-0.764; p = 0.004). The area under the ROC curve was determined to 0.817; 95% (CI) 0.732-0.901. CONCLUSION: Eye-tracking based measurements of gaze fixation and pupil reaction may be useful biomarkers of PD diagnosis. However, larger studies of eye-tracking parameters integrated into the screening of patients with suspected PD are necessary, to further investigate and confirm their diagnostic value.

Co-expression of ß Subunits with the Voltage-Gated Sodium Channel NaV1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics.

The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary ß2 and ß3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either ß2 or ß3 subunits in HEK-293 cells. The ß2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the ß2 subunits did not change the biophysical properties of the channel. In contrast, ß3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated ß3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the ß3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the ß3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of ß subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with ß2. The ß3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.

Identification of novel NaV1.7 antagonists using high throughput screening platforms.

Congenital Insensitivity to Pain (CIP) is a loss of function mutation resulting in a truncated NaV1.7 protein, suggesting a pivotal role in pain signaling and rendering it an important pharmaceutical target for multiple pain conditions. The structural homology in the NaV-channel family makes it challenging to design effective analgesic compounds without inducing for example cardiotoxicity or seizure liabilities. An additional approach to structural isoform selectivity is to identify compounds with use- or state-dependent profiles, i.e. inhibition efficacy based on the gating of the ion channel. In general nerve cells in damaged or inflamed tissue are more depolarized and electrically active compared to healthy nerve cells in for instance the heart. This observation has led to the design of two types of screening protocols emulating the voltage condition of peripheral neurons or cardiac tissue. The two voltage protocols have been developed to identify both use- and state-dependent antagonists. In this paper we describe an attempt to merge the two different protocols into one to increase screening efficacy, while retaining relevant state- and use-dependent pharmacology. The new protocol is constructed of two stimulation pulses and a slow voltage ramp for simultaneous assessment of resting and state-dependent block. By comparing all protocols we show that the new protocol indeed filter compounds for state-dependence and increase the prediction power of selecting use-dependent compounds.

Ion channel screening technology.

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.

Cellular HTS assays for pharmacological characterization of Na(V)1.7 modulators.

Ion channels are challenging targets in the early phases of the drug discovery process, especially because of the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The electrophysiological patch-clamp technique, which is the gold standard for studying ion channels, has low throughput and is not amenable to screening large numbers of compounds. However, for random high-throughput screening (HTS) of compounds against ion channel targets, a number of functional cellular assays have become available during the last few years. Here we use the sodium channel NaV1.7 stably expressed in human embryonic kidney 293 cells and compare three HTS assays-a Li flux atomic absorption spectroscopy (AAS) assay, a fluorescent imaging plate reader (FLIP, Molecular Devices, Sunnyvale, CA) membrane potential assay, and a fluorescence resonance energy transfer (FRET)-based membrane potential assay-to an automated electrophysiological assay (the Ionworks HT [Molecular Devices] platform) and characterize 11 known NaV inhibitors. Our results show that all three HTS assays are suitable for identification of NaV1.7 inhibitors, but as an HTS assay the Li-AAS assay is more robust with higher Z' values than the FLIPR and FRET-based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li-AAS assay regarding the potency of the NaV inhibitors investigated. This paper describes the first comparison between all the HTS assays available today to study voltage-gated NaVs, and the results suggest that the Li-AAS assay is more suited as a first HTS assay when starting an NaV drug discovery campaign.

3H-serotonin as a marker of oscillatory insulin secretion in clonal beta-cells (INS-1).

Serotonin release from preloaded pancreatic beta-cells has been used as a marker for insulin release in studying exocytosis from single cells using the amperometric technique. We found that single pancreatic beta-cells exhibited oscillations in exocytosis with a period of 1-1.5 min as measured amperometrically by serotonin release. We also show that 3H-serotonin can be used to monitor exocytosis from intact and streptolysin-O permeabilized clonal insulin-secreting cells preloaded with labeled serotonin and that serotonin release correlated with insulin secretion in the same cells. The use of 3H-serotonin provides a real-time indicator of exocytosis from populations of clonal insulin-secreting cells.

Distinct pharmacologic properties of neuromuscular blocking agents on human neuronal nicotinic acetylcholine receptors: a possible explanation for the train-of-four fade.

BACKGROUND: Nondepolarizing neuromuscular blocking agents (NMBAs) are extensively used in the practice of anesthesia and intensive care medicine. Their primary site of action is at the postsynaptic nicotinic acetylcholine receptor (nAChR) in the neuromuscular junction, but their action on neuronal nAChRs have not been fully evaluated. Furthermore, observed adverse effects of nondepolarizing NMBAs might originate from an interaction with neuronal nAChRs. The aim of this study was to examine the effect of clinically used nondepolarizing NMBAs on muscle and neuronal nAChR subtypes. METHODS: Xenopus laevis oocytes were injected with messenger RNA encoding for the subunits included in the human alpha1beta1epsilondelta, alpha3beta2, alpha3beta4, alpha4beta2, and alpha7 nAChR subtypes. The interactions between each of these nAChR subtypes and atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium were studied using an eight-channel two-electrode voltage clamp setup. Responses were measured as peak current and net charge. RESULTS: All nondepolarizing NMBAs inhibited both muscle and neuronal nAChRs. The neuronal nAChRs were reversibly and concentration-dependently inhibited in the low micromolar range. The mechanism (i.e., competitive vs. noncompetitive) of the block at the neuronal nAChRs was dependent both on subtype and the NMBA tested. The authors did not observe activation of the nAChR subtypes by any of the NMBAs tested. CONCLUSIONS: The authors conclude that nondepolarizing NMBAs concentration-dependently inhibit human neuronal nAChRs. The inhibition of the presynaptic alpha3beta2 nAChR subtype expressed at the motor nerve ending provides a possible molecular explanation for the tetanic and train-of-four fade seen during a nondepolarizing neuromuscular block.

Activation and inhibition of human muscular and neuronal nicotinic acetylcholine receptors by succinylcholine.

BACKGROUND: Succinylcholine is one of the most widely used muscle relaxants in clinical anesthesia and emergency medicine. Although the clinical advantages and cardiovascular side effects are well known, its mechanism of action within the human nicotinic cholinergic receptor system remains to be understood. The aim of this study was to investigate the effect of succinylcholine on human muscle and neuronal nicotinic acetylcholine receptor (nAChR) subtypes. METHODS: Xenopus laevis oocytes were injected with human messenger RNA for muscle and neuronal nAChR subunits. Receptor activation, desensitization, and inhibition induced by the natural ligand acetylcholine or by succinylcholine was studied using a multichannel two-electrode voltage clamp setup. Responses were measured as peak current and net charge. RESULTS: Succinylcholine concentration-dependently activated the muscle-type nAChR with an EC50 value of 10.8 microm (95% confidence interval, 9.8-11.9 microm), and after the initial activation, succinylcholine desensitized the muscle-type nAChR. Succinylcholine did not activate the neuronal nAChR subtypes alpha3beta2, alpha3beta4, alpha4beta2, or alpha7 at concentrations up to 1 mm and was a poor inhibitor at these receptor subtypes, with IC50 values above 100 microm. CONCLUSION: Succinylcholine activates the muscle-type nAChR followed by desensitization. The observation that succinylcholine does not inhibit the presynaptic alpha3beta2 autoreceptor at clinically relevant concentrations provides a possible mechanistic explanation for the typical lack of tetanic fade in succinylcholine-induced neuromuscular blockade. Finally, cardiovascular side effects (e.g., tachyarrhythmias) of succinylcholine are not mediated via direct activation of the autonomic ganglionic alpha3beta4 subtype because succinylcholine does not activate the neuronal nAChRs.

Ryanodine receptor-operated activation of TRP-like channels can trigger critical Ca2+ signaling events in pancreatic beta-cells.

There is little information available concerning the link between the ryanodine (RY) receptors and the downstream Ca(2+) signaling events in beta-cells. In fura-2 loaded INS-1E cells, activation of RY receptors by 9-methyl 5,7-dibromoeudistomin D (MBED) caused a rapid rise of [Ca(2+)]i followed by a plateau and repetitive [Ca(2+)]i spikes on the plateau. The [Ca(2+)]i plateau was abolished by omission of extracellular Ca(2+) and by SKF 96365. In the presence of SKF 96365, MBED produced a transient increase of [Ca(2+)]i, which was abolished by thapsigargin. Activation of RY receptors caused Ca(2+) entry even when the ER Ca(2+) pool was depleted by thapsigargin. The [Ca(2+)]i plateau was not inhibited by nimodipine or ruthenium red, but was inhibited by membrane depolarization, La(3+), Gd(3+), niflumic acid, and 2-aminoethoxydiphenyl borate, agents that inhibit the transient receptor potential channels. The [Ca(2+)]i spikes were inhibited by nimodipine and ryanodine, indicating that they were due to Ca(2+) influx through the voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR). Activation of RY receptors depolarized membrane potential as measured by patch clamp. Thus, activation of RY receptors leads to coherent changes in Ca(2+) signaling, which includes activation of TRP-like channels, membrane depolarization, activation of the voltage-gated Ca(2+) channels and CICR.

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

Growth hormone promotes Ca(2+)-induced Ca2+ release in insulin-secreting cells by ryanodine receptor tyrosine phosphorylation.

Elevation in cytoplasmic free Ca2+ concentration ([Ca2+]i) is a common mechanism in signaling events. An increased [Ca2+]i induced by GH, has been observed in relation to different cellular events. Little is known about the mechanism underlying the GH effect on Ca2+ handling. We have studied the molecular mechanisms underlying GH-induced rise in [Ca2+]i in BRIN-BD11 insulin-secreting cells. GH (500 ng/ml, 22 nm) induced a sustained increase in [Ca2+]i. The effect of GH on [Ca2+]i was prevented in the absence of extracellular Ca2+ and was inhibited by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+)-channel inhibitor nifedipine. However, GH failed to induce any changes in Ca2+ current and membrane potential, evaluated by patch-clamp recordings and by using voltage-sensitive dyes. When the intracellular Ca2+ pools had been depleted using the Ca(2+)-ATPase inhibitor thapsigargin, the effect of GH was inhibited. In addition, GH-stimulated rise in [Ca2+]i was completely abolished by ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and caffeine. GH induced tyrosine phosphorylation of ryanodine receptors. The effect of GH on [Ca2+]i was completely blocked by the tyrosine kinase inhibitors genistein and lavendustin A. Interestingly, treatment of the cells with GH significantly enhanced K(+)-induced rise in [Ca2+]i. Hence, GH-stimulated rise in [Ca2+]i is dependent on extracellular Ca2+ and is mediated by Ca(2+)-induced Ca2+ release. This process is mediated by tyrosine phosphorylation of ryanodine receptors and may play a crucial role in physiological Ca2+ handling in insulin-secreting cells.

Three-dimensional imaging of in situ specimens with low-dose electron tomography to analyze protein conformation.

We describe a novel three-dimensional (3-D) imaging tool for analysis of protein conformation of in situ samples. Sidec (Sidec Technologies AB, Stockholm, Sweden) electron tomography (SET) uses low-dose electron tomography and a refinement algorithm to reconstruct individual proteins and macromolecular complexes. The approach has successfully reconstructed therapeutic proteins in solution. In this study, we investigate the use of SET to visualize ion channels in cells and tissue samples. SET successfully resolved the volume and structural features of the target complex, showing that it was a tetrameric channel with a central pore. The technology could distinguish and provide 3-D images of the intra- and extracellular domains in the ion channel. In addition, SET was able to show that the channel associates in the form of a tetramer with the four subunits preorganized into dimers. While additional studies using smaller antibody markers are needed to resolve the subunit assembly further, this study demonstrates that SET is a valuable tool for visualization of in situ specimens and can provide important information on the subunit assembly of these macromolecular complexes, and thereby aid in the screening assay process in drug development.

Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose.

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic beta cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca(2+) concentrations, and intact sensitivity of the exocytotic machinery to Ca(2+). A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

Insulinotropic activity of the imidazoline derivative RX871024 in the diabetic GK rat.

The insulinotropic activity of the imidazoline derivative RX871024 was compared in pancreatic islets from nondiabetic Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats. RX871024 significantly stimulated insulin secretion in islets from both animal groups. The insulinotropic activity of RX871024 was higher than that of the sulfonylurea glibenclamide. This difference was more pronounced in islets from GK rats compared with Wistar rat islets. More importantly, RX871024 substantially improved glucose sensitivity in diabetic beta-cells, whereas glibenclamide stimulated insulin secretion about twofold over a broad range of glucose concentrations in nondiabetic and diabetic rats. RX871024 induced a faster increase in cytosolic free Ca(2+) concentration and faster inhibition of ATP-dependent K(+) channel activity in GK rat islets compared with Wistar rat islets. RX871024 also induced a more pronounced increase in diacylglycerol concentration in GK rat islets. These data support the idea that imidazoline compounds can form the basis for the development of novel drugs for treatment of type 2 diabetes, which can restore glucose sensitivity in diabetic beta-cells.