Renal Handling of Sclerostin in Response to Acute Glomerular Filtration Decline.

Deterioration of glomerular filtration rate (GFR) is associated with alterations of bone metabolism. It translates clinically to bone fragility and increased fractures rate among patients with impaired GFR. Recently, sclerostin (SCL) gained much attention as an important factor in pathogenesis of mineral and bone disturbances in patients with renal diseases. There is no data about SCL behaviour in patients with acute GFR decline. The aim of this study was to evaluate the renal handling of SCL. This is a prospective, single-centre observational study in patients undergoing nephrectomy due to urological indications. Serum and urinary SCL levels were measured prior and after nephrectomy. 25 patients were enrolled. After surgery, eGFR significantly declined (from 87.4±19.7 to 67.7±25.7 ml/min/1.73 m(2), p<0.0001). Nephrectomy caused more than 20 times higher renal fractional excretion of SCL [0.15 (interquartile range, IQR 0.09-0.40) vs. 2.78 (IQR 1.51-4.02)%, p<0.001], while its serum level remained intact [0.69 (IQR 0.57-0.90 vs. 0.65 (IQR 0.53-0.88) ng/ml, p=0.4]. The magnitude of eGFR reduction was associated inversely with change in urinary SCL fractional excretion (r=-0.6, p=0.001) and with alteration in serum SCL level (r=-0.5, p=0.01). Our results suggest that increased serum SCL concentrations at moderately reduced GFR are not due to diminished renal clearance. At more severely decreased GFR, elevated SCL concentration results from both increased production and reduced renal elimination.

Caloris Basin: An Enhanced Source for Potassium in Mercury's Atmosphere.

Enhanced abundances of neutral potassium (K) in the atmosphere of Mercury have been found above the longitude range containing Caloris Basin. Results of a large data set including six elongations of the planet between June 1986 and January 1988 show typical K column abundances of approximately 5.4 x 10(8) K atoms/cm(2). During the observing period in October 1987, when Caloris Basin was in view, the typical K column was approximately 2.7 x 10(9) K atoms/cm(2). Another large value (2.1 x 10(9) K atoms/cm(2)) was seen over the Caloris antipode in January 1988. This enhancement is consistent with an increased source of K from the well-fractured crust and regolith associated with this large impact basin. The phenomenon is localized because at most solar angles, thermal alkali atoms cannot move more than a few hundred kilometers from their source before being lost to ionization by solar ultraviolet radiation.

Characterization of defect levels in semi-insulating 6H-SiC by means of photoinduced transient spectroscopy and modulated photocurrent technique.

Parameters of electrically active defect centres in vanadium-doped 6H silicon carbide (6H-SiC:V) were investigated by means of the photoinduced transient spectroscopy (PITS) and modulated photocurrent (MPC) method. After a short description of the two techniques, experimental results are presented and briefly compared. Our aim is mainly to understand and explain these experimental results. In particular, in the PITS technique a shallow level seems to be at the origin of negative photoconductivity. Besides, in the same temperature range hole and electron levels can be detected at the same time. Finally, the detection of a given level seems to depend on the photon flux used to perform the PITS experiment. As far as the MPC experiment is concerned, it has put into evidence a very efficient shallow level. A numerical calculation was developed to simulate both experiments in order to understand the experimental results. By means of this simulation, we have explained all the phenomena observed experimentally in each technique and we propose a simple model for the distribution of electrically active defect centres in 6H-SiC:V crystals.

In utero and neonatal sensitivity of ApcMin/+ mice to radiation-induced intestinal neoplasia.

PURPOSE: To assess the sensitivity of ApcMin/+ mice (adenomatous polyposis coli Apc, multiple intestinal neoplasia, Min) to the development of intestinal adenomas after x-irradiation in utero, as neonates, or as young adults. MATERIALS AND METHODS: CHB6 ApcMin/+ mice were exposed to an acute dose of 2 Gy x-rays either in utero on day 7 or 14 post-conception, as 2-day or 10-day neonates or as 35-day young adults. Tumour identification and counting was performed 200-214 days later. RESULTS: Irradiation as 10-day-old neonates resulted in a significantly greater overall tumour incidence (average of about 130 tumours per animal) than irradiation as 35-day-old young adults (about 70 tumours). Irradiation as 2-day-old neonates resulted in an intermediate incidence (about 85 tumours). In contrast, the greatest tumour incidence observed after in utero irradiation of ApcMin/+ mice, of about 44 tumours per animal after 2 Gy irradiation at 14 days post-conception, was significantly lower than the incidence in irradiated adults. Tumour incidences after irradiation as 7-day embryos was not significantly raised above numbers in unirradiated controls (about 30 tumours). These tumour numbers include cystic crypts, largely radiation-induced, which were classed as early stage microadenomas on the basis of loss of wild-type Apc+ and expression of beta-catenin. CONCLUSIONS: The sensitivity of ApcMin/+ mice to the induction of intestinal tumours by radiation was shown to be in the order: 10 d neonates>2 d neonates>35 d young adults>14 d fetus>7 d embryo.

ATP increases Ca(2+)-activated K+ channel activity in isolated rat arterial smooth muscle cells.

Large conductance Ca(2+)-activated K+ (Kca) channels are known to be activated by phosphorylation through cAMP- and cGMP-dependent kinase activation. In pulmonary arterial smooth muscle KCa channels are directly activated by ATP (but not by non-hydrolysable analogues) independently of the presence of cyclic nucleotides or the catalytic subunits of protein kinases. This study was designed to determine whether direct activation of KCa channels by ATP is apparent in other types of arterial smooth muscle. KCa channels of similar conductance to those of rat pulmonary artery (approximately 250 pS) were found in membrane patches excised from isolated smooth muscle cells from rat aorta, mesenteric and basilar arteries. In myocytes isolated from each of these arteries, intracellular application of ATP (in the absence of exogenous cyclic nucleotides or catalytic subunits) reversibly increased the open state probability of KCa channels: a response markedly reduced by a specific inhibitor of protein kinase A. Nucleotide sequence analysis of KCa channels revealed no homology with the majority of protein kinases. It is concluded that phosphorylation of KCa channels through the activity of a membrane tethered kinase related to protein kinase A (but lacking its regulatory subunits) may play an important role in controlling K+ flux in a range of arterial smooth muscle cell types.

The role of cell contact and autostimulatory soluble factors in the proliferation of blast cells in acute myeloblastic leukemia.

The role of cellular interactions in stimulating leukemic cell proliferation was studied in AML. When peripheral blood blasts were cultured in suspension in flat-bottomed vessels at low cell numbers (15.6-500 x 10(3)/ml), the rate of DNA synthesis [( 3H]thymidine incorporation) was proportional to cell density. When cells were cultured under otherwise identical conditions in round-bottomed vessels, to induce cell crowding, the rate of DNA synthesis was independent of cell numbers and was significantly augmented (p less than or equal to 0.01). Physical separation of cells in round-bottomed (crowded) cultures by latex beads reduced DNA synthesis to the same rate as that in flat-bottomed (non-crowded) cultures. In contrast, addition of mitomycin-treated autologous blasts had little effect in crowded cultures but increased DNA synthesis in non-crowded cultures. DNA synthesis was also enhanced: by reducing culture volume to concentrate any diffusible factors produced by the cells or by blast cell conditioned medium from autologous blasts grown under crowded conditions. Cells cultured in suspension for 72 hr under crowded/non-crowded conditions both formed blast cell colonies in methyl cellulose; however, the number of colonies derived from crowded cultures was greater. These results indicate that cell proximity promotes proliferation of blasts and blast cell progenitors; both close cell contact and autostimulatory soluble factors are important in regulating growth of AML blasts.

Endogenous interleukin-1 can regulate the autonomous growth of the blast cells of acute myeloblastic leukemia by inducing autocrine secretion of GM-CSF.

We have studied peripheral blood blast cells from a patient with acute myeloblastic leukemia (AML) whose cells proliferated autonomously at high cell density, but only in the presence of adherent cells. At low cell density in suspension culture and in a clonogenic assay, blast cell growth was stimulated by recombinant granulocyte macrophage colony stimulating factor (GM-CSF) and recombinant interleukin-1 (IL-1) independently. The response to rIL-1 was inhibited (less than 90%) by anti-GM-CSF, suggesting that the proliferative response to IL-1 was mediated by GM-CSF. This was supported by experiments which demonstrated that blast cell conditioned medium prepared in the presence of IL-1 contained GM-CSF activity which stimulated the growth of normal granulocyte-macrophage precursors (CFU-GM) and of homologous GM-CSF responsive AML blasts. As IL-1 but no GM-CSF activity was detected in BCCM prepared without exogenous IL-1 and a neutralizing antibody to IL-1 inhibited the autonomous growth of blasts in suspension culture, we conclude that the endogenous secretion of IL-1 by leukemic cells stimulated autocrine GM-CSF secretion, inducing autonomous growth of the blast cell population.

Role of autocrine and paracrine production of granulocyte-macrophage colony-stimulating factor and interleukin-1 beta in the autonomous growth of acute myeloblastic leukaemia cells--studies using purified CD34-positive cells.

The blast cells from some patients with acute myeloblastic leukemia (AML) proliferate autonomously in vitro. We have previously identified four groups of AML blasts based upon their growth characteristics in vitro, in particular the degree of autonomous growth. We have now measured the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-1 beta (IL-1 beta) by AML cells with different growth characteristics, using two sensitive enzyme-linked immunosorbent assays. Our results show a correlation between the capacity of AML blasts to produce GM-CSF and IL-1 beta and the ability to grow autonomously in vitro. Blasts from cells with no autonomous growth (n = 5) secreted low or undetectable amounts of GM-CSF and IL-1 beta. Blasts with totally autonomous growth (n = 10) secreted the highest levels of GM-CSF (mean 2469 pg/10(3) cells) and IL-1 beta (mean 3156 pg/10(6) cells). Whereas blasts with partially autonomous growth (n = 9) secreted intermediate levels of GM-CSF (mean 270 pg/10(6) cells) and IL-1 beta (mean 931 pg/10(6) cells). In order to determine whether GM-CSF production was autocrine or the consequence of paracrine secretion by differentiated leukemic cells, we studied the degree of autonomous growth and production of GM-CSF by CD34-positive blasts from eight patients whose unfractionated cells produced GM-CSF. We found that CD34-positive blasts from six of these cases grew autonomously to a degree comparable to that of the unfractionated cells, and that CD34-positive blasts produced GM-CSF either autonomously or in response to recombinant IL-1 beta. Our data suggests that in the majority of cases, CD34-positive blasts are capable of autonomous growth and autocrine GM-CSF production, however this is variably regulated by the paracrine production of IL-1 beta by CD34-negative cells.

Evidence for internal autocrine regulation of growth in acute myeloblastic leukemia cells.

Blast cells from 70% of patients with acute myeloid leukemia (AML) show some evidence of in vitro autonomous growth, which appears to be related to the autocrine secretion of growth factors, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF). In the majority of cases, the growth factors appear to be involved in classical extracellular autocrine or paracrine loops with neutralizing antibodies to the relevant cytokine inhibiting growth. In a minority, however, antibodies do not inhibit growth despite evidence of secretion of the cytokine. There is evidence for intracellular autocrine loops in murine leukemic cell lines. In this study, we wished to investigate for the presence of such intracellular loops involving GM-CSF in AML blast cells. Blast cells from 11 patients with AML were cultured in the presence of either neutralizing GM-CSF antibody or an antisense oligonucleotide directed against GM-CSF. We also studied the effect of the oligonucleotide on the autonomous growth of cells whose production of GM-CSF had been apparently abolished by either interleukin-1 receptor antagonist (IL-1Ra) or following blast cell purification using the CD34 antigen. The autonomous growth of the blast cells from nine of the 11 patients was inhibited by the antisense oligonucleotide (but not by the control sense oligonucleotide). However, only six of the nine were inhibited by the anti-GM-CSF antibody. Similarly, in one patient whose CD34 purified blast cells continued to show a high degree of autonomous growth but did not produce detectable GM-CSF, growth was inhibited by the antisense oligonucleotide but not by antibody, while in another patient whose cells were inhibited by IL-1Ra with, again, loss of detectable GM-CSF, growth could be further inhibited by the addition of the oligonucleotide but not the antibody. These studies provide evidence that intracellular autocrine loops involving GM-CSF are involved in the autonomous growth of some AML blast cells.

Ion channels, oxygen sensation and signal transduction in pulmonary arterial smooth muscle.

In contrast to systemic arteries those of the pulmonary circulation constrict in response to hypoxia: a mechanism known as "hypoxic pulmonary vasoconstriction" (HPV). It is has been established that changes in alveolar oxygen tension are the primary stimulus for triggering HPV. The mechanisms underlying this unique response are unknown. Neither the role of the endothelium nor that of the smooth muscle of the pulmonary arteries in oxygen sensing has been elucidated, although both tissues may be involved. Evidence is accumulating to suggest that K+ channels located in the plasma membranes of pulmonary arterial smooth muscle cells may play an important role in oxygen sensing. This process may involve novel mechanisms of signal transduction and K+ channel regulation by charged intermediates, redox reactions or membrane-delimited factors.

The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity.

Adenosine triphosphate (ATP) bioluminescence was used to determine whether there was a linear relationship between cultured cell number and measured luminescence using the luciferin-luciferase reaction. In all the cells tested including peripheral blood mononuclear cells (MNC), MOLT-4, HL-60, TF-1, NFS-60 and L-929 cell lines there was a significant correlation as determined by Spearman's rank correlation coefficient (p > 0.00001). These observations were then used to determine whether ATP bioluminescence could be used as a suitable substitute for tritiated thymidine uptake as a measure of cell proliferation. The cell lines MOLT-4, HL-60, TF-1 and NFS-60 showed a strong correlation between thymidine uptake and ATP bioluminescence (p > 0.00001 for all cell types). Additionally the ATP method could detect the cytokine dependent proliferation on TF-1 and NFS-60 cells by granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) respectively. The tumour necrosis factor alpha (TNF)-induced cytotoxic effect on L-929 cells could also be accurately detected using this method. It would therefore appear to be possible to use ATP bioluminescence in the detection of cytokine activity in a number of different bioassays.

Outcome of first-trimester exposure to low-dose methotrexate in eight patients with rheumatic disease.

PURPOSE: Methotrexate (MTX), when used to treat malignancy or psoriasis, has been implicated in anecdotal reports as a teratogen or abortifacient in the first trimester of pregnancy. We are unaware of any previous reports that describe the course of gestation and the effect on subsequent offspring in patients treated with low-dose oral MTX for rheumatoid arthritis, and therefore present our experience. PATIENTS AND METHODS: We report on eight women experiencing 10 pregnancies. Mean number of weeks of gestation while taking MTX was 7.5 (range 2 to 20 weeks). Outcome of pregnancies included five full-term babies (FTB), three spontaneous abortions (SAB), and two elective abortions. RESULTS: There were no significant differences in either the FTB or SAB group when considering risk factors including smoking, alcohol, concomitant medications, and age. One of three in the SAB group had recurrent abortions prior to MTX therapy. All five of the FTB group had uncomplicated pregnancies and deliveries. All offspring were of normal height and weight at birth with no physical abnormalities. All children reached growth, development, and intellectual stages normally, and their present mean age is 11.5 years. No observed learning disabilities or medical abnormalities have occurred in any of these children. CONCLUSION: In this uncontrolled study we failed to demonstrate tertogenicity of MTX. However, the possibility of abortion due to MTX use remains.

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.

Barbiturates inhibit ATP-K+ channels and voltage-activated currents in CRI-G1 insulin-secreting cells.

1. Patch-clamp recording techniques were used to examine the effects of barbiturates upon the ATP-K+ channel, and voltage-activated channels present in the plasma membrane of CRI-G1 insulin-secreting cells. 2. Thiopentone inhibited ATP-K+ channel activity when applied to cell-attached patches or the intracellular or extracellular surface of cell-free patches. Secobarbitone and pentobarbitone were also effective inhibitors of ATP-K+ channels in cell-free patches, whereas phenobarbitone was ineffective. 3. The diabetogenic agent, alloxan, which is structurally related to the barbiturates also produced an inhibition of ATP-K+ channel activity in outside-out patches. 4. Whole-cell ATP-K+ currents were used to quantify the effects of the barbiturates: concentration-inhibition curves for thiopentone, secobarbitone and pentobarbitone resulted in IC50 values of 62, 250 and 360 microM respectively. Phenobarbitone at a concentration of 1 mM was virtually ineffective. 5. Calculation of the apparent membrane concentrations for these drugs indicate that for a given degree of ATP-K+ channel inhibition a similar concentration of each barbiturate is present in the membrane. This suggests that hydrophobicity plays a primary role in their mechanism of action. The pH-dependence and additive nature of barbiturate block also indicates a membrane site of action. 6. Thiopentone, (100 microM) was also found to inhibit differentially voltage-activated whole-cell currents. The relative potency of thiopentone at this concentration was 0.64, 0.38 and 0.12 for inhibiting Ca2+, K+ and Na+ currents respectively when compared with its ability to inhibit the ATP-K+ channel.

Nucleotide-dependent activation of KATP channels by diazoxide in CRI-G1 insulin-secreting cells.

1. Patch-clamp recording techniques were used, to examine the effects of diazoxide on KATP currents in CRI-G1 insulin-secreting cells in the presence of non-hydrolysable nucleotides. 2. In the presence of non- or slowly-hydrolyzed ATP analogues, bathing the intracellular aspect of cell-free membrane patches diazoxide inhibited KATP channel activity. 3. Under whole-cell recording conditions, with various non-hydrolysable nucleotides present intracellularly (after dialysis), diazoxide induced KATP current activation. The largest activation occurred with Mg-adenylyl-(beta, gamma-methylene) diphosphate (Mg-AMP-PCP) present in the dialysing solution. This activation was diazoxide- and nucleotide-concentration-dependent. 4. In the absence of Mg2+, or in the presence of manganese (Mn2+) ions intracellularly, diazoxide did not induce KATP current activation, regardless of the species of nucleotide present in the pipette. 5. Intracellularly applied trypsin prevented the activation of KATP currents by diazoxide in the presence of Mg-AMP-PCP, an effect reversed by co-application of intracellular polymethylsulphonyl fluoride with the trypsin. 6. The application, by dialysis, of a CRI-G1 cell lysate, with negligible Mg-ATP, resulted in a substantial activation of the KATP current by diazoxide. 7. It is concluded that diazoxide can activate KATP channel currents by two separate pathways, one requiring a phosphorylation process, the other the presence of an intracellular protein coupled with a Mg-purine nucleotide.

Electrophysiological effects of endothelin-1 and their relationship to contraction in rat renal arterial smooth muscle.

The electophysiological effects of endothelin-1 (ET-1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch-clamp measurements using freshly isolated smooth muscle cells from this tissue. Pharmacological experiments revealed that ET-1 produced constriction of renal arteries dependent on the influx of extracellular Ca(2+), mediated solely through ET(A) receptor stimulation. Current-clamp experiments revealed that renal arterial myocytes had a resting membrane potential of approximately 32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET-1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly. Under voltage-clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage-ramps revealed an outward current activated at approximately -30 mV, sensitive to both 4-AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K(+) (K(V)) and Ca(2+)-activated K(+) (BK(Ca)) channels. Under voltage-clamp, ET-1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ET(A) receptor stimulation. Thus, in conclusion, activation of ET(A) receptors by ET-1 causes inhibition of K(V) and BK(Ca) channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L-type Ca(2+) channel activity providing an influx pathway for extracellular Ca(2+) essential for contraction.

A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes.

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.

Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.

1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.

Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line.

1. The effects of diazoxide on ATP-K+ channel currents, recorded from the insulin-secreting cell line, CRI-G1, were studied using patch-clamp techniques. 2. Under current-clamp recording conditions diazoxide (0.6 mM), inhibited action potential activity and hyperpolarized CRI-G1 cells with a concomitant increase in membrane conductance. Recordings from voltage-clamped whole-cells and isolated patches indicate that activation of ATP-K+ channel currents underlie these effects. 3. Diazoxide elicited an activation of ATP-K+ channels which had been partially inhibited by ATP, on application to either surface of the plasma membrane, although it was more effective when applied directly to the cytoplasmic side. Activation of the ATP-K+ currents involves an increase in the single channel open-state probability and an apparent increase in the number of functional channels. 4. Activation was observed only when Mg-ATP was present in the cytoplasmic bathing solution. There was no activation of currents by diazoxide when ATP, in the absence of Mg2+ ions, or Mg-AMP-PNP was present to inhibit the ATP-K+ channels. 5. In the absence of ATP and Mg2+ ions in the cytoplasmic bathing solution, diazoxide (0.6 mM) produced an inhibition of ATP-K+ currents. 6. Cromakalim (BRL 34915) at 10 microM and 100 microM had no significant effects on ATP-K+ currents. 7. It is concluded that diazoxide-induced activation of ATP-K+ channel currents probably involves phosphorylation of the channel or some closely associated membrane protein.

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line.

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.