Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells

Abstract

Hyperpolarization of patch-perforated GH3 rat anterior pituitary cells in high-K+ Ca(2+)-free medium reveals an inwardly rectifying K+ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs+, Ba2+, Cd2+ and Co2+, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH3 cell inwardly rectifying K+ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.

Okadaic acid and calyculin A enhance the effect of thyrotropin‐releasing hormone on GH3 rat anterior pituitary cells excitability

“…This effect does not seem to be due to a non specific membrane effect of OKA since: (i) no effect of inhibitor was detected before treatment of cells with TRH; (it) similar effects to those of OKA are observed with CL-A; (iii) the phase I response to TRH was not impaired by treatment of cells with the inhibitors; (iv)-~la--O?f tncubation times and inhibitc~r concentrations as low as 2 nM are enough to elicit the effects of OKA; and (v) even at the highest concentration of OKA used in this study (100 nM), the enhancement of TRH effects is selectively exerted on inward rectifying K + currents (see [13]). It has been previously suggested that inhibition of an inwardly rectifying current by TRH constitutes an important factor regulating the resting potential and conductance underlying increased frequency of APs induced by the hormone in GH~ cells [12,13]. Recent experiments from our laboratory indicated that treatment of patch-perfo-.~ 4,.~dl r~.L.i " "…”

“…We have recently shown that electrical responses to TRH are fully preserved when patch-perforated current-clamped GHa cells are studied [5]. On the other hand, recent experiments from our laboratory indicated that treatment of GH3 cells with OKA specifically potentiates the reductions caused by TRH on inward rectifying K ÷ currents [13]. In this report, the effects of OKA and CL-A on electrical activity of the cells are explored.…”

“…Other Materials and Methods were identical to those described in [5,13]. AP's frequency was determined in periods of at least 80 s either before or 2-5 min after the start of the indicated treatments.…”

“…The mechanisms and conductance pathways responsible for the increased production of APs are not well known. It has been suggested that inhibition of various outward currents, including the transient outward current named IK~ and IK(~) [7][8][9], the voltage-and Ca2+-activated K ÷ curret~t [5,10,11], or the inwardly rectifying K + current [12,13], is the cause of the second phase of hyperexcitability. It has also been suggested that phosphorylation by protein kinase C (PKC) is involved in modulation of the K ÷ channel activity which determines the firing rate [1,2,4,14,15].…”

“…Our results indicate that a phosphorylation--dephosphorylation mechanism may be involved in the regulation of the second phase of TRH action. Furthermore, the possibility that a type 2A protein phosphatase plays a major role in regulating the delayed effects of TRH via its effect on the inward rectifying K" current [12,13] is discussed.…”