Patch-clamp Whole-cell Current Recording Research Articles

Role of Ca2+ in pancreatic cell death induced by alcohol metabolites

Whereas alcohol itself, even in high concentrations, has little effect on the functional performance of isolated pancreatic acinar cells, non-oxidative metabolites (fatty acid ethyl esters [FAEE] and fatty acids [FA]) can cause Ca(2+)-dependent necrosis. The mechanism of action of FAEE has been investigated using a combination of patch clamp whole-cell current recording and Ca(2+) imaging. At low stimulation intensities, FAEE evoke repetitive short-lasting cytosolic Ca(2+) spikes, which are inhibited by caffeine, used as an inositol trisphosphate receptor antagonist. With more intense stimulation, sustained elevations of the cytosolic Ca(2+) concentration are observed, which can be prevented by pharmacological inhibition of the conversion of FAEE to FA. It is therefore the FA and not the FAEE that cause necrosis. The effect of FA cannot be blocked by inositol trisphosphate receptor antagonists. Fatty acids elicit a marked reduction in the cytosolic adenosine triphosphate (ATP) level. The patch clamp experiments show that the toxic sustained Ca(2+) signal generation induced by FA can be prevented by adding ATP to the cell interior. The toxic alcohol effects are principally due to FAEE produced under non-oxidative conditions and their subsequent conversion to FA. The FA-induced necrosis is Ca(2+)-dependent. The destructive sustained Ca(2+) signals are due to inhibition of mitochondrial function with failure of ATP generation.

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet beta-cells.

Intracellular cAMP-dependent modulation of L-type Ca2+ channel activation in cultured rat islet beta-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca2+ current (ICa) showed a fast activation followed by a slow inactivation, and was sensitive to Ca2+ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak ICa in a concentration-dependent manner. Values of the half-activation potentials (V1/2), taken from activation curves for ICa, were -16.7 +/- 1.8 and -21.9 +/- 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet beta-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca2+ channels.

Ca 2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca 2+ signals, since they are morphologically polarized and generate agonist-specific Ca 2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca 2+ signals and the Ca 2+-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca 2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca 2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimick these responses. The shortlasting local Ca 2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Acetylcholine- and caffeine-evoked repetitive transient Ca(2+)-activated K+ and C1- currents in mouse submandibular cells.

1. Resting and acetylcholine-induced membrane currents were measured in single mouse submandibular acinar cells using the patch-clamp whole-cell current recording technique. 2. Micromolar ACh activated a large, sustained outward, Ca(2+)-dependent K+ current and a single transient inward Ca(2+)-dependent C1-current. 3. Nanomolar ACh induced a series of transients in both the K+ and C1- currents; C1- current activation was now observed throughout the period of agonist application. We consider this repetitive transient current activation better able to support sustained fluid and electrolyte secretion than the response elicited by a high dose of agonist. 4. Repetitive K+ and C1- current transients were also induced by 1 mM-caffeine, consistent with caffeine-induced Ca2+ release from the Ca(2+)-sensitive Ca2+ stores which are thought to comprise part of the pathway for activation of secretion. 5. The ACh-induced current transients were inhibited by 10 mM-caffeine, 100 microM-IBMX and 10 microM membrane-permeable cyclic AMP. Therefore, it seems likely that caffeine is able to inhibit agonist-induced calcium mobilization via a cyclic AMP-dependent pathway.

Stimulatory and inhibitory actions of VIP and cyclic AMP on cytoplasmic Ca2+ signal generation in pancreatic acinar cells.

In pancreatic acinar cells the effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic AMP (dbcAMP) alone and during stimulation with acetylcholine (ACh) or internally applied inositol trisphosphate (InsP3) were investigated utilizing the patch-clamp whole-cell current recording configuration to assess changes in cytoplasmic Ca2+ concentration by measurement of Ca2+ dependent Cl- current. VIP (1 nM) and dbcAMP (0.5 mM) each evoked repetitive pulses of Ca(2+)-dependent Cl- current. A high VIP concentration (100 nM) acutely and reversibly inhibited the responses evoked by 1 nM VIP and also acutely inhibited ACh-evoked responses. DbcAMP (2 mM) also reversibly inhibited the ACh-evoked responses. Neither VIP nor dbcAMP were able to inhibit InsP3-evoked repetitive Ca2+ pulses. VIP in a low concentration can generate cytosolic Ca2+ oscillations via cyclic AMP, but high VIP concentrations inhibit both ACh and VIP-evoked Ca2+ signals and this effect is mediated by high intracellular cyclic AMP levels.

Does inositol tetrakisphosphate play a role in the receptor-mediated control of calcium mobilization?

The evidence for and against an important role for inositol 1,3,4,5 tetrakisphosphate (Ins 1,3,4,5 P 4) in receptor-mediated Ca 2+ mobilization is reviewed. Data obtained from patch-clamp whole-cell current recording studies on internally perfused exocrine acinar cells show that the acetylcholine (ACh)-evoked sustained increase in Ca 2+-dependent K + current caused by an increase in [Ca 2+] i cannot be mimicked by internal application of inositol 1,4,5-trisphosphate (Ins 1,4,5 P 3), but only by a combination of Ins 1,4,5 P 3 and Ins 1,3,4,5 P 4. The sustained response evoked by Ins 1,4,5 P 3 + Ins 1,3,4,5 P 4 is dependent on the presence of external Ca 2+ as is the effect of ACh. Only those inositol trisphosphates able to evoke Ca 2+ release from internal stores can support the action of Ins 1,3,4,5 P 4 in evoking responses that are acutely dependent on extracellular Ca 2+ (Ca 2+ influx). The various arguments presented against an involvement of Ins 1,3,4,5 P 4 are discussed. The main point emerging is that most studies are inadequately controlled and it is concluded that there is a strong need for whole-cell current recording studies combined with pipette fluid exchange to be carried out in many more systems. The major problem in this field is that the precise site and mechanism of action of Ins 1,3,4,5 P 4 are unknown and that the pathway for Ca 2+ uptake during receptor activation is inadequately defined.