Kisspeptin regulates gonadotroph and somatotroph function in nonhuman primate pituitary via common and distinct signaling mechanisms

Abstract

Kisspeptins (Kps) have emerged as key players in the control of reproductive-axis function, in which they operate as primary regulators of hypothalamic GnRH release. In addition, recent data indicate that Kps can also directly act on the pituitary to stimulate LH and GH release in primary pituitary cell culture prepared from rats, cows, and sheep. We present herein evidence that Kps (specifically Kp-10) can also stimulate LH and GH release in primary pituitary cell cultures prepared from female baboons (Papio anubis), a species that more closely models human physiology. The stimulatory effect of Kp-10 on LH and GH release was dose and time dependent and enhanced the hormonal responses to their major regulators (GnRH for LH; GHRH/ghrelin for GH) without affecting the release of other pituitary hormones (TSH, FSH, ACTH, prolactin). Use of pharmacological intracellular signaling blockers indicated Kp-10 signals through phospholipase C, protein kinase C, MAPK, and intracellular Ca(2+) mobilization, but not adenylyl cyclase, protein kinase A, extracellular Ca(2+) influx (through L-type channels), or nitric oxide synthase, to stimulate both LH and GH release. Interestingly, blockade of mammalian target of rapamycin or phosphoinositol 3-kinase activity fully abolished the stimulatory effect of Kp-10 on LH but not GH release. Of note, estradiol enhanced the relative LH response to Kp-10, alone or in combination with GnRH. In sum, our data are the first to provide evidence that, in a primate model, there is a functional Kp-signaling system within the pituitary, which is dynamically regulated and may contribute to the direct control of gonadotropic and somatotropic axes.

Fig. 1.

Direct actions of Kp-10 (10 nm) on baboon LH and GH synthesis and secretion. A, Effect of 4 h treatment with Kp on LH (A1) and GH (A2) release. B, Time-dependent effect of Kp on LH (B1) and GH (B2) release. C, Time-dependent effect of Kp on LH (C1) and GH (C2) mRNA levels. Data are expressed as percent of control (set at 100%) at 4 h (A) or 30 min (B and C). Values represent the mean ± sem (n = 4 individual experiments, three to four wells/experiment). Values that do not share a common letter (a, b, c, d) are statistically different. Asterisks indicate values that significantly differ from their respective control values (same incubation time period). *, P < 0.05; **, P < 0.01.

Fig. 2.

Interaction of Kp-10 (10 nm) with regulators of gonadotrope and somatotrope function in primary pituitary cell cultures from baboons. A, Effect of 4 h treatment of Kp and/or GnRH (10 nm) on LH secretion. B, Effect of 4 h treatment of Kp and/or GHRH, ghrelin (10 nm), or SST (100 nm) on GH secretion. Values are expressed as percentage of controls, set at 100% within each experiment, and represent the mean ± sem of four independent experiments (three to four wells/experiment). Values that do not share a common letter (a, b, c, d) differ significantly (P < 0.05).

Fig. 3.

Intracellular signaling pathways of Kp-10-stimulated baboon LH and GH release. Effect of the inhibition of AC (MDL-12,330A; 10 μm), protein kinase A (H89; 15 μm), PLC (U73122; 50 μm), protein kinase C (Go6983; 20 μm), MAPK (PD-98,059; 10 μm), extracellular Ca2+ L-type channels (nifedipine; 1 μm), intracellular Ca2+ channels (thapsigargin; 10 μm), NOS (L-arginine methyl ester hydrochloride; 10 μm), mTOR (rapamycin; 10 nm), and PI3K (wortmannin; 1 μm) on kisspeptin-stimulated LH (A) and GH (B) release. On the day of the experiment, inhibitors were added to the incubation media 90 min before Kp treatment (4 h; 10 nm). Values are expressed as percentage of vehicle-treated controls without inhibitor (set at 100%) within each experiment, and represent the mean ± sem of three to five independent experiments (three to four wells/treatment per experiment). Values that do not share a common letter (a or b) significantly differ (P < 0.05).

Fig. 4.

Interaction of Kp-10 (10 nm) with regulators of gonadotrope and somatotrope function in the absence or presence of estradiol (−E2 or +E2, respectively; 10 nm) in primary pituitary cell cultures from baboons. A, Effect of 4 h treatment of Kp and/or GnRH (10 nm) on LH secretion. B, Effect of 4 h treatment of Kp and/or GHRH, ghrelin (10 nm), or SST (100 nm) on GH secretion. Data are expressed as percentage of controls without E2 (set at 100%), and represent the mean ± sem of three to four independent experiments (three to four wells per experiment). Values that do not share a common letter (a, b, c, d) are statistically different. Asterisks indicate values that significantly differ from their respective control values (same treatment in the absence of estradiol). *, P < 0.05; **, P < 0.01.