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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 insulinsecreting 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 40pM and 0.2pm, respectively. 3 Diazoxide (0.6-1.0mM) 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 (20mM), 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 17pM and 27nm, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6 Diazoxide (0.6mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 pM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7 Neither tolbutamide (1.0mM) nor glibenclamide (10 M) 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.

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-Gi 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 1OpM and 100pM 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.

Uridine nucleotides are known to cause constriction of pulmonary arterial smooth muscle. However, the P2 receptor subtypes underlying the contractile effects of these nucleotides in the pulmonary circulation have not been determined. We have used myography and the patch-clamp recording technique to compare the effects of UTP and UDP in isolated small pulmonary arteries (diameter 100 to 400 microm) and their constituent smooth muscle cells. In endothelium-denuded arteries, both UTP and UDP (0.01 to 3 mmol/L) induced concentration-dependent increases in tension that were independent of P2X receptor stimulation. The UDP-mediated increase in tension was significantly less sensitive to the nonselective P2 receptor blocker suramin than the UTP-mediated increase in tension. In single isolated arterial myocytes, voltage-clamped at -50 mV (close to the resting membrane potential of these cells), application of both UTP and UDP evoked periodic oscillations of inward current primarily because of a Ca2+-activated Cl- current (ICl,Ca). Oscillations of ICl,Ca evoked by UTP were reversibly inhibited by suramin, although those evoked by UDP were insensitive to the antagonist. In addition to confirming the presence of classical P2Y2 receptors, these results also provide functional evidence for the existence of a novel UDP receptor in pulmonary arterial myocytes, which may contribute to pyrimidine-evoked vasoconstriction. This notion is supported by molecular evidence that demonstrates the presence of P2Y6 receptor transcripts in rat pulmonary arterial smooth muscle.

Understanding the way in which single nucleotide polymorphisms and mutations in the human genome result in individual susceptibility to disease is a major goal in the postgenomic era. Such knowledge should accelerate the development of personalised medicine in which drug treatment can specifically match an individual's genotype. High-throughput DNA sequencing is generating the initial information required, but new technologies are required that can rapidly characterise the phenotypic effects of the identified polymorphisms. For example, many thousands of allelic variants of the p53 gene have been described and are responsible for more than 50% of cancers, however few of the protein products have been functionally characterised. Here we have quantified in parallel the effects of mutations and polymorphisms on the DNA-binding function of the p53 oncoprotein using a protein microarray, allowing their subclassification according to functional effect. Protein-protein interactions between p53 variants and (i) a regulatory oncoprotein, (ii) a regulatory kinase resulting in on-chip phosphorylation, are also described, suggesting the more general utility of this high-throughput assay format.

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ATP-sensitive K + -channel currents were recorded from isolated mem­brane patches and voltage-clamped CRI-G1 insulin-secreting cells. Inter­nal Mg 2+ ions inhibited ATP-K + channels by a voltage-dependent block of the channel current and decrease of open-state probability. The run­ down of ATP-K + channel activity was also shown to be [Mg 2+ ] i depen­dent, being almost abolished in Mg 2+ -free conditions. Substitution of Mn 2+ for Mg 2+ did not prevent run-down, nor did the presence of phos­phate-donating nucleotides, a protease or phosphatase inhibitor or replacement of Cl by gluconate.

A novel class of Ca(2+)-activated K+ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these "KCa,ATP" channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of KCa,ATP channels to voltage, intracellular Ca2+ ([Ca2+]i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K+ and are approximately 20 times more selective for K+ ions than Na+ ions, with a K+ permeability (PK) of 4.6 x 10(-13) cm s-1.Ca2+ ions applied to the intracellular membrane surface of KCa,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca2+ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of KCa,ATP channels by Mg-ATP has an EC50 of approximately 50 microM. Although the EC50 is unaffected by [Ca2+]i, channel activation by Mg-ATP is enhanced by increasing [Ca2+]i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of KCa,ATP channels to Ca2+. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of KCa,ATP channels to Ca2+ and therefore voltage is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)