Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx.

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 µM tolbutamide and 20 µM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 µM) or the L-type Ca(2+) channel blocker nicardipine (2 µM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 µM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 µM but not 1 µM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 µM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 µM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 µM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 µM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

Local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices.

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority. Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9'A mice (1) and α6 L9'S mice (2), allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices. In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.