High extracellular Ca2+ stimulates Ca2+-activated Cl- currents in frog parathyroid cells through the mediation of arachidonic acid cascade.

Elevation of extracellular Ca(2+) concentration induces intracellular Ca(2+) signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(2+) pathways, but the direct mechanism responsible for the rise of intracellular Ca(2+) concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(2+) signaling in frog parathyroid cells and show that Ca(2+)-activated Cl(-) channels are activated by intracellular Ca(2+) increase through an inositol 1,4,5-trisphophate (IP(3))-independent pathway. High extracellular Ca(2+) induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50) ∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(2+)-induced and Ca(2+) dialysis-induced currents reversed at the equilibrium potential of Cl(-) and were inhibited by niflumic acid (a specific blocker of Ca(2+)-activated Cl(-) channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(2+)-induced current, suggesting the change of intracellular Cl(-) concentration in a few minutes. Extracellular Ca(2+)-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca(2+)-induced current. IP(3) dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca(2+)-induced conductance. These results indicate that high extracellular Ca(2+) raises intracellular Ca(2+) concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(-) conductance.

A calcium-receptor agonist induces gustatory neural responses in bullfrogs.

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC(50) for NPS R-467 was about 20 microM. Cross-adaptation experiments were performed to examine the effect of 30 microM NPS R-467 and 100 microM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 microM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 microM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 microM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K(+) conductance decrease.

Biophysical properties of voltage-gated Na+ channels in frog parathyroid cells and their modulation by cannabinoids.

The membrane properties of isolated frog parathyroid cells were studied using perforated and conventional whole-cell patch-clamp techniques. Frog parathyroid cells displayed transient inward currents in response to depolarizing pulses from a holding potential of -84 mV. We analyzed the biophysical properties of the inward currents. The inward currents disappeared by the replacement of external Na+ with NMDG+ and were reversibly inhibited by 3 micromol l-1 TTX, indicating that the currents occur through the TTX-sensitive voltage-gated Na+ channels. Current density elicited by a voltage step from -84 mV to -24 mV was -80 pA pF-1 in perforated mode and -55 pA pF-1 in conventional mode. Current density was decreased to -12 pA pF-1 by internal GTPgammaS (0.5 mmol l-1), but not affected by internal GDPbetaS (1 mmol l-1). The voltage of half-maximum (V1/2) activation was -46 mV in both perforated and conventional modes. V1/2 of inactivation was -80 mV in perforated mode and -86 mV in conventional mode. Internal GTPgammaS (0.5 mmol l-1) shifted the V1/2 for activation to -36 mV and for inactivation to -98 mV. A putative endocannabinoid, 2-arachidonoylglycerol ether (2-AG ether, 50 micromol l-1) and a cannabinomimetic aminoalkylindole, WIN 55,212-2 (10 micromol l-1) also greatly reduced the Na+ current and shifted the V1/2 for activation and inactivation. The results suggest that the Na+ currents in frog parathyroid cells can be modulated by cannabinoids via a G protein-dependent mechanism.

Serotonin differentially modulates the electrical properties of different subsets of taste receptor cells in bullfrog.

Serotonin (5-hydroxytryptamin, 5-HT) is localized in taste bud cells of vertebrates. Effects of the external application of 5-HT on the membrane currents of frog taste receptor cells (TRCs) were investigated using patch-clamp technique in whole-cell configuration. The 5-HT (0.1-1 micro m) and 5-HT1A receptor agonist (+/-)-8-OH-2-(D1-n-propyl-amino)tetralin (8-OH-DPAT) (1-20 micro m) inhibited both voltage-gated sodium current (INa) and voltage-gated potassium current (IK) in 50% of TRCs, but potentiated IK without any significant effect on INa in another subset of 18% of TRCs. Voltage-gated currents in the residual TRCs were not affected by 5-HT or 8-OH-DPAT. External application of 10 micro m forskolin and 300 micro m 8-cpt cAMP [8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate] mimicked the inhibitory effect of 5-HT and 8-OH-DPAT on IK and INa while internal dialysis with 50 micro m protein kinase A inhibitor prevented the 5-HT-mediated inhibitory effects on IK and INa in TRCs. Internal dialysis of TRCs with high Ca2+-pipette solution (1 micro m) increased the IK in 58% of TRCs. The 5-HT reversibly increased the [Ca2+]i in 17% of TRCs when measured by Ca2+-imaging using a Ca2+-sensitive dye (fura-2 AM). These results suggest that 5-HT differentially modulates the voltage-gated membrane currents in different subsets of TRCs.