Simultaneous recordings of cytosolic Ca²⁺ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH₄C₁ clonal cells was studied using fura-2 to monitor the cytosolic Ca²⁺ level ([Ca²⁺](i)) in single cells, combined with recordings of membrane potential and current. The average value of [Ca²⁺](i) was 109 nM (mean ± SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca²⁺](i), peaking at 795 ± 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca²⁺-activated K⁺ channels. In 75% of the cells the initial peak in [Ca²⁺](i) was followed by a prolonged plateau phase at 247 ± 76 nM (n = 84). In current clamp the second-phase elevation of [Ca²⁺](i) was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization silent cells. This elevation of [Ca²⁺](i) was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca²⁺](i) was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca²⁺-free conditions eliminated the second-phase elevation in [Ca²⁺](i), whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca²⁺](i) in the GH₄C₁ cells. The second-phase elevation of [Ca²⁺](i) caused by thyroliberin is the result of influx through voltage-sensitive Ca²⁺ channels, without involving agonist-gated channels.