Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110ß in the regulation of insulin secretion from rodent and human beta cells.

AIMS/HYPOTHESIS: Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and ß (p110α and -ß) in insulin granule recruitment and exocytosis in rodent and human islets. METHODS: The p110α and p110ß subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. RESULTS: Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85ß) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110ß blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kß (p110ß/p85ß) even when p110ß catalytic activity was inhibited. Conversely, both the wild-type p110ß and a catalytically inactive mutant directly facilitated exocytosis. CONCLUSIONS/INTERPRETATION: Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110ß is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

AIMS/HYPOTHESIS: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS: Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS: Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION: Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

The pharmacokinetics of propofol in laboratory animals.

1. The pharmacokinetics of propofol in an emulsion formulation ('Diprivan') have been studied after single bolus doses to rats, dogs, rabbits and pigs, and after single and multiple infusions to dogs. Venous blood propofol concentrations were determined by h.p.l.c. with u.v. or fluorescence detection. Curve fitting was performed using ELSFIT. 2. The distribution of propofol in blood and its plasma protein binding have been studied in rat, dog, rabbit and man. Protein binding was high (96-98%), and in most species propofol showed appreciable association with the formed elements of blood. 3. Where an adequate sampling period was employed the pharmacokinetics of propofol were best described by a three-compartment open 'mammillary' model. Propofol was distributed into a large initial volume (1-21/kg) and extensively redistributed (Vss = 2-10 x body weight) in all species. Clearance of propofol by all species was rapid, ranging from about 30-80 ml/kg per min in rats, dogs and pigs to about 340 ml/kg per min in rabbits.

The pharmacokinetics of Casodex in laboratory animals.

1. The pharmacokinetics of Casodex, a novel, non-steroidal antiandrogen, have been investigated following single oral and i.v. doses and during daily oral dosing to male and female rats and male dogs. 2. The binding of 14C-Casodex to rat, dog and human plasma proteins, determined by equilibrium dialysis, was high with values greater than 95%; in dog there was evidence for decreased binding at concentrations greater than 12 micrograms/ml. 3. Casodex was slowly absorbed over prolonged periods and its bioavailability decreased with increase in dose from 72% and 88% in male and female rats respectively at 1 mg/kg to 10% and 12% at 250 mg/kg; in dog bioavailability decreased from 100% at 0.1 mg/kg to 31% at 100 mg/kg. 4. Elimination of Casodex from plasma was slow with terminal elimination half-lives of about 1 day in rat and about 6 days in dog. On daily administration to rats Casodex accumulates slightly in plasma at 10 mg/kg but not at 250 mg/kg; in dog appreciable accumulation (9-12-fold), calculated from the ratio of trough plasma concentrations at steady state to those after a single dose, was observed at 2.5 and 10 mg/kg, but at 100 mg/kg the accumulation ratio was much lower (4-fold).

Pharmacokinetics and protein binding of propofol in patients with cirrhosis.

The pharmacokinetics and protein binding of propofol were studied in ten patients with cirrhosis and in ten control patients undergoing elective surgery. All patients received 2.5 mg.kg-1 propofol as an intravenous bolus injection for the induction of anesthesia. Whole blood propofol concentrations were measured at intervals up to 12 h, using a high-performance liquid chromatography (HPLC) technique. Propofol protein binding was estimated by equilibrium dialysis 10 min after injection of propofol. Individual propofol profiles for all patients were best described by a three-compartment open mammillary model. Rapid and slow propofol distribution half-times were observed, followed by an elimination phase with a half-time of 4-5 h. Propofol total body clearance was reduced (1.99 +/- 0.68 l.min-1) in the patients with cirrhosis but did not differ significantly from that in the control patients (2.30 +/- 0.61 l.min-1). The apparent volume of distribution at steady state (Vdss) was similar in the two groups. No significant difference in elimination half-life was observed between the two groups. Propofol was extensively bound (mean: 97-98%) to the plasma protein of both cirrhotic and control groups. This study shows that propofol pharmacokinetics and protein binding of propofol following a single intravenous bolus dose were not markedly affected by uncomplicated cirrhosis of the liver.

Lithium determined in serum with an ion-selective electrode.

We evaluated the performance of the lithium ion-selective electrode (ISE) in the Du Pont Na/K/Li analyzer. Lithium concentrations in 106 serum samples from patients being treated with lithium were measured in duplicate with the ISE and by flame photometry. The slope of the regression line for the two methods was 1.004 with a standard error of the estimate of 0.049 mmol/L (x = flame photometry, y = ISE). Lithium measurements by the ISE method in serum or aqueous standards were linear to greater than 2.0 mmol/L. Within-run CVs for low (0.31 mmol/L) and high (1.15 mmol/L) lithium controls were 5.9% and 1.7%, respectively (n = 20). Day-to-day CVs for the same controls were 9.8% and 3.3%, respectively (n = 20). There was no significant interference when the concentrations of sodium, potassium, calcium, or magnesium were varied, nor did intervening urinary lithium analyses affect the measurement of serum lithium. Results for lithium measurement in four serum-based survey materials compared well with results by isotope dilution/mass spectrometry.