Free radical-mediated tolbutamide desensitization of K+ATP channels in rat pancreatic beta-cells.

To study the effects of hydroxyl radicals on the sensitivity of the ATP-sensitive K+ (K+ ATP) channel to tolbutamide, we used patch clamp and microfluorometric techniques in pancreatic beta-cells isolated from rats. cell-attached membrane patches, exposure of the cells to 0.3 mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8 mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8 microM before and immediately after exposure to H2O2. After prolonged exposure (>20 min) to H2O2, the IC50 was increased to 15 microM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1 mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10 microM tolbutamide. Addition of 0.3 mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10 min, followed by a sustained pronounced increase in [Ca2]i. After more than 20 min of exposure of cells to 0.3mM H2O2, [Ca2]i was increased to above 2 microM. Treatment of the cytoplasmic face of inside-out membrane patches with 1 microM Ca2+ attenuated the tolbutamide-sensitivity of the K+ATP channel, but not the ATP-sensitivity of the channel. These findings indicate that H2O2 reduces tolbutamide sensitivity by inducing a sustained increase in [Ca2+]i.

PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells.

The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells.

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.

Control of catecholamine release and blood pressure with octreotide in a patient with pheochromocytoma: a case report with in vitro studies.

A 65-year-old male patient with pheochromocytoma, whose hypertensive episodes were uncontrolled using conventional therapy, was successfully treated with octreotide (SMS 201-995). The serum catecholamine level and the urinary excretion of catecholamines decreased after 300 microgram/day of octreotide was administered. To clarify the mechanisms of octreotide that lower catecholamine released from a tumor, we studied the in vitro effects of octreotide on membrane potentials and voltage-dependent Ca(2+) channel (VDCC) current using the whole-cell patch-clamp technique in single pheochromocytoma cells dispersed after tumor resection. The action potentials were reversibly inhibited with 10 microM octreotide. In addition, the VDCC current evoked by depolarized pulses from the holding potential of -60 mV was inhibited with 10 microM octreotide. Octreotide is useful for controlling blood pressure before surgery in some patients with uncontrolled hypertension caused by a pheochromocytoma.

P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells.

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.

A case of renal juxtaglomerular cell tumor: usefulness of segmental sampling to prove autonomic secretion of the tumor.

A 27-year-old female patient had been treated for hypertension with conventional therapy for years, because renal vein renin levels failed to show lateralization in renal venous samplings and a renal juxtaglomerular cell tumor (RJGCT) had gone undiagnosed. Abdominal computed tomography revealed a mass at the middle of the right kidney. The right renal venogram demonstrated distinct segmental veins from the upper pole and from the middle and lower poles in the right kidney. On segmental renin sampling from each renal vein, the plasma renin concentration (PRC) of the segmental veins from the middle and lower poles was higher than that from other sites. We diagnosed RJGCT of the right kidney and performed right-sided nephrectomy. After the resection, the PRC rapidly decreased. Immunohistochemical studies using antihuman renin antibodies revealed positive staining of the tumor cells. It is an important strategy to make a segmental sampling at the site as close as possible to the RJGCT.

[Ca2+]i-reducing action of cAMP in rat pancreatic beta-cells: involvement of thapsigargin-sensitive stores.

In the present study, we examined the ability of adenosine 3',5'-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. [Ca2+]i and reduced pyridine nucleotide, NAD(P)H, were measured in rat single beta-cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+]i elevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 microM), and an incretin glucagon-like peptide-1-(7-36) amide (10(-9) M), as well as by glucose (16.7 mM). The [Ca2+]i-reducing effects of cAMP were greater at elevated glucose (8.3-16.7 mM) than a basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+]i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10-100 nM also reduced sustained [Ca2+]i elevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in beta-cells. [Ca2+]i-reducing effects of cAMP were inhibited by 0.3 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+]i-reducing effects of cAMP were not altered by ryanodine, an ER Ca(2+)-release inhibitor, Na(+)-free conditions, or diazoxide, an ATP-sensitive K+ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca2+]i elevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of beta-cell metabolism. Together with the known [Ca2+]i-increasing action of cAMP, our results reveal dual regulation of beta-cell [Ca2+]i by the cAMP-signaling pathway and by a physiological incretin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP-38 or PACAP-27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP-38 and PACAP-27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single beta-cells, monitored by dual-wavelength fura-2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in beta-cells. PACAP antiserum also neutralized the [Ca2+]i-increasing action of synthetic PACAP-38 and PACAP-27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca2+]i in beta-cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from beta-cells, increased [Ca2+]i in beta-cells at both and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP-38 were detected in islets and a beta-cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7. [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive beta-cells, many of which were recruited by PACAP-38 into [Ca2+]i responses. 8. These results indicate that PACAP-38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses beta-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.

Current status of PACAP as a regulator of insulin secretion in pancreatic islets.

PACAP-27 and PACAP-38 as low as 10(-13) M stimulate insulin release from rat islets in a glucose-dependent manner. PACAP also glucose dependently increases cAMP and [Ca2+]i in rat islet beta cells. The [Ca2+]i and insulin secretory responses to PACAP exhibit a similar concentration-response relationship, exhibiting a peak at 10(-13) M. When the [Ca2+]i response is abolished by nitrendipine, a blocker of L-type Ca2+ channels, the insulin response is also inhibited. Insulinotropic peptides glucagon, GLP-1, and VIP also increase [Ca2+]i in beta cells, but only in the nanomolar concentration range. PACAP is 4 logs more potent that VIP, a peptide that exhibits 68% amino acid homology and shares the type II PACAP receptor with PACAP. Immunoreactivity for the type I PACAP receptor is demonstrated in rat islets. Furthermore, PACAP immunoreactivity is demonstrated in nerve fibers and islets in rat pancreas. Based on these findings, we can draw the following conclusions: (1) PACAP is localized in pancreatic nerve fibers and islets; (2) PACAP in the subpicomolar range stimulates insulin release from islets; (3) the stimulation of insulin release is mediated by the cAMP-dependent increase in [Ca2+]i in beta cells; (4) all the PACAP effects are glucose-dependent; (5) PACAP is the most potent insulinotropic hormone known, and (6) the type I PACAP receptor appears to mediate the action of PACAP in the subpicomolar range. Finally, we hypothesize that PACAP is a pancreatic peptide of both neural and islet origin and functions as an intrinsic potentiator of glucose-induced insulin secretion in pancreatic islets (FIG 6).

cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells.

cAMP and the insulinotropic peptides that raise cAMP glucose-dependently increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells, which is tightly linked to the potentiation of glucose-induced insulin release. We examined whether cAMP increases [Ca2+]i in specific cooperation only with glucose or also with other insulin secretagogues that act through different mechanisms. [Ca2+]i in single rat pancreatic beta-cells was measured by dual-wavelength fura-2 microfluorometry. In the presence of a stimulatory concentration of glucose (8.3 mmol/l) and the moderate elevation in [Ca2+]i induced by it, forskolin, an activator of adenylyl cyclase, or dibutyryl cAMP produced a marked additional increase in [Ca2+]i but was ineffective at the basal 2.8 mmol/l glucose. These cAMP-elevating agents also potentiated the effect of tolbutamide on [Ca2+]i. The cAMP-induced increase in [Ca2+]i was completely and selectively inhibited by a blocker of cAMP-dependent protein kinase A (PKA), and by nitrendipine, a blocker of the L-type Ca2+ channel. However, in the presence of high KCl and the [Ca2+]i elevation induced by it, a rise in cAMP failed to further increase [Ca2+]i, whereas BAY K8644, an agonist of L-type Ca2+ channels, evoked an additional increase in [Ca2+]i. Under low Na+ conditions, the [Ca2+]i response to cAMP was observed in the majority of the cells. In the cells in which glucose at 4.5-5 mmol/l was inadequate to increase [Ca2+]i, the glucose together with a rise in cAMP often increased [Ca2+]i. Likewise, tolbutamide and a rise in cAMP acted in concert to increase [Ca2+]i. Thus, cAMP left-shifted the concentration-[Ca2+]i response relationship for glucose and tolbutamide. In conclusion, the cAMP-PKA pathway acts in selective synergism with glucose and tolbutamide to initiate [Ca2+]i signals in pancreatic beta- cells. cAMP appears to regulate beta-cell sensitivity to glucose and tolbutamide. In contrast, cAMP fails to cooperate with high KCl to increase [Ca2+]i. It is suggested that cAMP acts mainly on a site that is more proximal but functionally linked to the L-type Ca2+ channel, thereby finally increasing Ca2+ influx through this channel.

Two distinct modes of Ca2+ signalling by ACh in rat pancreatic beta-cells: concentration, glucose dependence and Ca2+ origin.

1. Calcium signalling by acetylcholine (ACh) in single rat pancreatic beta-cells was studied. The cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual-wavelength fura-2 microfluorometry. 2. In the presence of basal glucose (2.8 mM), 10(-6) to 10(-4) M ACh (high ACh) transiently increased [Ca2+]i. The [Ca2+]i response to 10(-5) M ACh was little altered under Ca(2+)-free conditions. Brief pulses of 10(-5) M ACh evoked successive [Ca2+]i responses, which were progressively inhibited by 0.2-0.5 microM thapsigargin, a specific inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. 3. Elevation of glucose to 8.3 mM, a concentration which stimulates insulin release, increased [Ca2+]i to an initial peak followed by a sustained, moderate elevation. Addition of 10(-8) to 10(-7) M ACh (low ACh) evoked a further increase in [Ca2+]i. The [Ca2+]i response to 10(-7) M ACh was completely inhibited under Ca(2+)-free conditions by 1 microM nitrendipine, a blocker of L-type Ca2+ channels, and by 100 microM diazoxide, an opener of ATP-sensitive K+ channels. 4. In the presence of 8.3 mM glucose, [Ca2+]i responses to 10(-5) M ACh were reduced but not abolished by Ca(2+)-free conditions, nitrendipine and diazoxide. Successive [Ca2+]i transients induced by 10(-5) M ACh pulses in the presence of nitrendipine were progressively inhibited by thapsigargin. 5. The results revealed two distinct modes of Ca2+ signalling: low ACh increases [Ca2+]i by stimulating Ca2+ influx through voltage-dependent L-type Ca2+ channels only in the beta-cells in which glucose has already elevated [Ca2+]i, while high ACh increases [Ca2+]i at basal as well as stimulatory glucose concentrations by releasing Ca2+ from the ER. The former mechanism is likely to relate to the potentiator action and the latter to the initiator action of ACh on insulin release. High ACh and elevated glucose provoke both modes of Ca2+ signalling.

Effects of thapsigargin, an intracellular CA2+ pump inhibitor, on insulin release by rat pancreatic B-cell.

This is the first report as to the effects of thapsigargin (Tg), an inhibitor of intracellular Ca2+ pumps, on insulin release by pancreatic B-cells. Tg does not alter basal insulin release by the isolated islets, with 3 mM glucose. However, it potentiates high glucose-induced insulin release: potentiation of the first phase response is dose-related in a concentration range of 1.3-40 microM. In isolated B-cells, Tg causes a minimal rise in basal cytosolic free calcium concentration ([Ca2+]i) and eliminates high glucose-induced initial lowering of [Ca2+]i. Tg does not alter glucose oxidation by the islets and the islet insulin content. An elimination of glucose-induced sequestration of Ca2+ into Tg-sensitive intracellular pool(s) is considered to be the cause of Tg potentiation of glucose effect on insulin release.

[Molecular mechanism of the K+ATP channel in pancreatic beta-cells].

The ATP-sensitive K+ (K+ATP) channel plays a key role in secretion of insulin in response to glucose-stimulation in pancreatic beta-cells. Inhibition of the channel does not require hydrolysis of ATP and results from a direct binding of ATP4- to the channel. MgADP relieves the channel inhibition by ATP by decreasing affinity of the channel to ATP. We suggest two-sites model regarding channel modulations by these nucleotides; one is the ATP-inhibition site which is bound by ATP4-, and the other the modulation site, which is bound by MgADP and thereby decreases the sensitivity of the channel to ATP. Sulphonylureas-binding sites may be different from these nucleotide-binding sites described above.