Growth hormone‐releasing hormone triggers pacemaker activity and persistent Ca2+ oscillations in rat somatotrophs.

Abstract

1. The effects of brief applications of growth hormone-releasing hormone (GHRH) to male rat somatotrophs in culture were analysed with the perforated patch clamp technique to record changes in potential or with fura-2 imaging techniques to measure variations of cytosolic Ca2+ concentration ([Ca2+]1). 2. Silent somatotrophs (n = 61) had a mean resting potential of -37 + 1 mV and a mean basal [Ca2+]. of 30 + 4 nM. Brief GHRH applications (30 nM, 40 s) triggered rhythmic action potentials (23-6 + 0 9 mV, 613 + 82 ms, 0X21 + 0-02 Hz) and [Ca2+], increase (to 352 + 30 nM) followed by rhythmic [Ca2+]i transients (to 138 + 6 nM) that persisted up to 90 min after the last GHRH application. Both action potentials and [Ca2+], transients were totally and reversibly blocked by removing external Ca2P or Nae or by adding inorganic Ca2+ channel blockers or nifedipine (3 uM).3. Somatostatin (1-300 nM), carbamylcholine (0 1-1 /M) and muscarine (0 1-1 jM) each had a dose-dependent inhibitory effect, from a decrease of Ca2+ spike duration and frequency to a complete block of the GHRH-evoked action potentials. 4. The present results show that somatotrophs in culture have intrinsic membrane properties that allow them to sustain a pacemaker activity and subsequent long-lasting sequences of[Ca2+], oscillations triggered by short pulses of GHRH and inhibited by somatostatin and muscarinic agonists.Pulsatile growth hormone (GH) secretion by somatotrophs is a complex process that includes control by at least two hypothalamic factors with opposite actions, the GH-releasing hormone (GHRH) and somatostatin, the GH-releaseinhibiting hormone (see review by Bertherat, Bluet-Pajot & Epelbaum, 1995). In addition, a variety of substances, including neurotransmitters, neuropeptides, growth factors and cytokines, endogenous to the anterior pituitary gland, can act as local modulators of somatotroph activity in a paracrine or autocrine manner (Houben & Denef, 1990). In the male rat, GH release is characterized by a striking regular ultradian rhythm composed of GH peaks of 1 h duration every 3-4 h (Tannenbaum & Martin, 1976). In vitro, brief (4-10 min) GHRH applications evoke a longlasting GH secretion in perifused anterior pituitary cells (Sheppard, Moor & Kraicer, 1985;Kato & Suzuki, 1986; Weiss, Cronin & Thorner, 1987). For example, 10 min pulses of GHRH (3 nM) every 3 h each evoke a 1 h rise in GH secretion (Weiss et al. 1987). Studies with intracellular and patch clamp recording techniques of the effects of GHRH application in vitro have provided contradictory results; GHRH evokes either a small and transient depolarization (Chen, Israel & Vincent, 1989a;Naumov, Herrington & Hille, 1994) perfused at a rate of 2-5 ml min-'. The Na+-free solution was A prepared by substituting choline chloride or tris(hydroxymethyl) aminomethane (Tris) for NaCl in the standard solution. The low-Clsolution was prepared by substituting isethionic acid (sodium salt)for NaCl in the standard solution. The Ca2+-free solution was prepared by adding 1 mm EGTA to a Ca2+...

Expression of Ca2+-Mobilizing EndothelinAReceptors and Their Role in the Control of Ca2+Influx and Growth Hormone Secretion in Pituitary Somatotrophs

“…GHRH activates its seven membrane domain receptors that are coupled positively to adenylyl cyclase. The increase in cAMP stimulates tetrodotoxin-insensitive pacemaker current to depolarize the membrane and initiate L-type Ca 2ϩ channel-driven AP firing and the associated [Ca 2ϩ ] i transients (Lussier et al, 1991a;Kwiecien et al, 1997). In contrast, somatostatin receptors are coupled negatively to adenylyl cyclase (Reisine and Bell, 1995), and their activation hyperpolarizes the cells to abolish GHRH-induced APs and [Ca 2ϩ ] i transients (Mollard et al, 1988;Lussier et al, 1991c;Sims et al, 1991).…”

Characterization of a Plasma Membrane Calcium Oscillator in Rat Pituitary Somatotrophs

“…High amplitude spontaneous [Ca 2ϩ ] i transients are not unique for somatotrophs, as immortalized pituitary cells also exhibit such changes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) influx in spontaneous and GHRH-controlled electrical activity in somatotrophs (19) and GC somatotroph lines (3).…”

Differential Management of Ca²⁺ Oscillations by Anterior Pituitary Cells: A Comparative Overview

“…The same procedure does not affect the first phase of the response to TRH but suppresses the potentiated activity of the second phase and the [Ca 2+ ] i plateau, showing that they result from Ca 2+ entry [8](not shown). In somatotropes action potentials and [Ca 2+ ] i transients are abolished by removal of external Ca 2+ [12, 14, 15, 26, 28](fig. 4b), by Co 2+ (2–5 m M ) [11, 12, 14], or treatment with verapamil (100 µ M ) [14], high doses of Ni 2+ (5 m M ) [11]or nifedipine (1–3 µ M ) [28].…”

“…It should be stressed that, in some cells, action potentials and [Ca 2+ ] i transients, once evoked, persist during the whole recording session (1–2 h) even after removal of GHRH [28]. In spontaneously active somatotropes, GHRH increases the amplitude and the frequency of spontaneous action potentials [9]and [Ca 2+ ] i transients [26].…”

“…In silent somatotropes, focal applications of GHRH (10–30 n M for 30–40 s) trigger a long-lasting sequence of action potentials [28]and induce a 10-fold increase in [Ca 2+ ] i followed by rhythmic [Ca 2+ ] i transients in cells not subjected to patch clamping. It should be stressed that, in some cells, action potentials and [Ca 2+ ] i transients, once evoked, persist during the whole recording session (1–2 h) even after removal of GHRH [28].…”