Biomedical subjects

C Schwanstecher

Publications and source records attributed to C Schwanstecher.

22 records · Page 2Linked to original sources

PubMed1992

Diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic beta-cells.

1. In mouse pancreatic beta-cells the regulation of the diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel (K-ATP-channel) was examined by use of the patch-clamp technique. 2. In intact beta-cells incubated at 37 degrees C in the presence of 3 mM D-glucose, diazoxide did not affect the single channel conductance but stimulated channel-opening activity. Diazoxide produced half-maximal effects at 82 microM and 13 fold activation at maximally effective concentrations (300-400 microM). The response to diazoxide (300 microM) was not completely suppressed by saturating tolbutamide concentrations (1 or 5 mM). 3. Inside-out patch-clamp experiments were carried out using an experimental protocol favouring phosphorylation of membrane proteins. Under these conditions diazoxide was ineffective in the absence of any nucleotides, weakly effective in the presence of MgATP (26 or 87 microM) and strongly effective in the presence of the Mg complexes of adenosine 5'-diphosphate, 2'-deoxyadenosine 5'-diphosphate or guanosine 5'-diphosphate (MgADP, MgdADP or MgGDP). 4. In inside-out patches exposed to nucleotide-free solutions, saturating concentrations of tolbutamide did not cause complete block of K-ATP-channels. When the channels were activated by MgdADP (48 microM), tolbutamide was even less effective. Sensitization of MgdADP-induced channel activation by diazoxide further weakened the effects of tolbutamide. 5. Diazoxide (50 or 300 microM) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2+ and ADP (1 mM). 6. In the presence of Mg2", the K-ATP-channel-blocking potency of cytosolic ATP decreased in the order inside-out> outside-out> whole-cell configuration of the patch-clamp technique.7. It is concluded that the K-ATP-channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.

Adenine Nucleotides↗

PubMed1992

Cytosolic nucleotides enhance the tolbutamide sensitivity of the ATP-dependent K+ channel in mouse pancreatic B cells by their combined actions at inhibitory and stimulatory receptors.

In the plasma membrane of pancreatic B cells, a K+ channel (K-ATP channel) has been identified that is regulated by cytoplasmic nucleotides. This channel is inhibited by sulfonylureas. We have previously shown that the potency of tolbutamide is much lower in excised membrane patches than in intact cells, unless the internal side of the membrane is exposed to the Mg2+ complex of ADP (MgADP). In the present study, the mechanism of this interactive control by sulfonylureas and nucleotides was examined using the inside-out configuration of the patch-clamp technique. When test solutions containing Mg2+ ions were applied, the opening activity of the K-ATP-channels was strongly stimulated by 2'-deoxyadenosine-5'-diphosphate (dADP) or GDP, slightly stimulated by ADP, and inhibited by adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) or adenylyl-imidodiphosphate (AMP-PNP). In the presence of Mg2+, not only ADP but also its analogues dADP (1 mM) and ADP beta S (0.1 mM) enhanced the potency of tolbutamide for channel inhibition; dADP at a low concentration (0.2 mM), GDP (0.2-1 mM), and AMP-PNP (0.2 mM) did not alter the potency of tolbutamide. The particular feature of the test solutions that enhanced the potency of tolbutamide was the presence of Mg(2+)-bound and free nucleotides at channel-stimulating and channel-inhibiting concentrations, respectively. In the presence of Mg2+ and 0.2 mM dADP or 0.2-1 mM GDP, 0.2 mM AMP-PNP intensified the response to tolbutamide by serving as channel-inhibiting component. MgAMP-PNP did not stimulate the opening activity of the K-ATP channel. The sensitivity to tolbutamide that was enhanced by a submaximally effective ADP concentration was further increased by AMP-PNP or ATP but not by GDP. The sensitivity to the sulfonylurea analogue meglitinide was also enhanced by ADP. It is concluded that nucleotides inhibit and activate the K-ATP channel by interaction with two separate receptor sites at the cytoplasmic face of the B cell membrane. Effective inhibition of the channel openings by sulfonylureas results from the simultaneous occupation of both sites by appropriate nucleotides.

Adenine Nucleotides↗

PubMed1990

Tolbutamide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic B-cells.

The patch-clamp technique was used to examine the tolbutamide-sensitivity of the adenosine 5'-triphosphate (ATP)-dependent K+ channel in mouse pancreatic B-cells. When studied at 37 degrees C in cell-attached membrane patches, this channel had a single-channel conductance of 88 pS and was half-maximally inhibited by 2.2 mumol/l tolbutamide in the presence of 3 mmol/l D-glucose and 10 mumol/l nifedipine. The tolbutamide-induced decrease in the amplitude of the single-channel currents indicated that the membrane potential was sufficiently depolarized for initiation of insulin release by 30 but not by 10 mumol/l of tolbutamide. Using 300 mumol/l diazoxide to open the ATP-dependent K+ channels already closed by 3 mmol/l D-glucose alone, it was demonstrated that initiation of insulin release requires closure of more than 98% of all ATP-dependent K+ channels. In excised inside-out membrane patches, the K+ channel-blocking potency of tolbutamide was maximally enhanced by 0.3 mmol/l adenosine 5'-diphosphate (ADP) at the cytoplasmic side. This ADP effect required the presence of Mg2+. Inhibition of K+ channel activity by ATP, ADP (Mg2(+)-free) or their non-hydrolyzable analogues adenylyl-imidodiphosphate (AMP-PNP) and alpha, beta methylene adenosine 5'-diphosphate (AMP-CP) was not accompanied by enhancement of tolbutamide-sensitivity. The results suggest that cytosolic MgADP controls tolbutamide-sensitivity by interaction with a receptor site not identical with the site mediating channel closure and that this control plays a role in the intact B-cell.

Action Potentials↗

PubMedDate not supplied

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity.

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.

Action Potentials↗