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Reduction of an inwardly rectifying K+ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH3 rat anterior pituitary cells. However, the existence of an inwardly rectifying K+ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH3 cells. In this report, an inwardly rectifying component of K+ current is indeed demonstrated in perforated-patch voltage-clamped GH3 cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K+ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K+ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K+ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH3 cells.

Treatment of rat anterior pituitary GH3 cells with caffeine causes a reversible enhancement of electrical activity superimposed over a depolarization of the plasma membrane potential. Similar results are obtained with theophylline, but not with isobutylmethylxanthine or forskolin. The effects of caffeine are not related to Ca2+ liberation from intracellular stores since they are not affected by incubation of the cells with ryanodine or thapsigargin. Furthermore, caffeine-induced hyperpolarization of the membrane is not detectable even in cells in which Ca2+ liberation from inositol 1,4,5-trisphosphate-sensitive compartments produces a prominent transient hyperpolarization in response to thyrotropin-releasing hormone. Reductions of Ca2+-dependent K+ currents caused by partial block of L-type Ca2+ channels by caffeine are not sufficient to explain the effects of the xanthine, since the results obtained with caffeine are not mimicked by direct blockade of Ca2+ channels with nisoldipine. GH3 cell inwardly rectifying K+ currents are inhibited by caffeine. Studies on the voltage dependence of the caffeine-induced effects indicate a close correlation between alterations of electrical parameters and reported values of steady-state voltage dependence of inactivation of these currents. We conclude that, as previously shown for thyrotropin-releasing hormone, modulation of inwardly rectifying K+ currents plays a major role determining the firing rate of GH3 cells and its enhancement by caffeine.

Exposure of GH3 rat anterior pituitary cells to cholera toxin for 2-4 h significantly increased the thyrotropin-releasing-hormone(TRH)-induced inhibition of the inwardly rectifying K+ current studied in patch-perforated voltage-clamped cells. On the other hand, the current reduction became almost totally irreversible after washout of the neuropeptide. Comparison of the effects elicited by the toxin with those of 8-(4-chlorophenylthio)-cAMP or forskolin plus isobutylmethylxanthine indicated that, although the irreversibility may be due, at least in part, to elevations of cAMP levels, the enhancement of the TRH-induced inhibition of the current is not mediated by the cyclic nucleotide. Only reductions on the inwardly rectifying K+ current, but not those elicited by TRH on voltage-dependent Ca2+ currents, were increased by the treatment with cholera toxin. In current-clamped cells showing similar rates of firing, the second phase of enhanced action-potential frequency induced by TRH was also significantly potentiated by cholera toxin. Measurements of [Ca2+]i oscillations associated with electrical activity, using video imaging with fura-2-loaded cells, demonstrated that cholera toxin treatment causes a clear reduction of spontaneous [Ca2+]i oscillations. However, this did not prevent the stimulatory effect of TRH on oscillations due to the action potentials. In cholera-toxin-treated cells, the steady-state, voltage dependence of inactivation of the inward rectifier was shifted by nearly 20 mV to more negative values. These data suggest that the inwardly rectifying K+ current plays an important role in maintenance of the resting K+ conductance in GH3 cells. Furthermore, the TRH-induced reductions on this current may be an important factor contributing to the increased cell excitability promoted by the neuropeptide.

Thyrotropin-releasing hormone (TRH) causes a transient hyperpolarization followed by several minutes of increased action potential frequency in patch-perforated current-clamped GH3 cells. Treatment of cells for 5 min with either 2 or 100 nM of the protein phosphatase inhibitor okadaic acid does not affect electrical activity of the cells, but potentiates the enhancement of action potential frequency elicited by a subsequent addition of TRH. Alternatively, 100 nM (but not 2 nM) of okadaic acid added during the second phase of TRH action, further increases the frequency of firing above that produced by the hormone. Similar effects to those of 2 nM okadaic acid are observed with 20 nM calyculin A. These data suggest that a protein phosphatase plays a major role in regulating the delayed effects of TRH on cell excitability in GH3 cells.