Multiple Pathways for High Voltage‐Activated Ca²⁺ Influx in Anterior Pituitary Lactotrophs and Somatotrophs

Abstract

The present study demonstrates that a significant proportion of high voltage-activated (HVA) Ca(2+) influx in native rat anterior pituitary cells is carried through non-L-type Ca(2+) channels. Using whole-cell patch-clamp recordings and specific Ca(2+) channel toxin blockers, we show that approximately 35% of the HVA Ca(2+) influx in somatotrophs and lactotrophs is carried through Ca(v) 2.1, Ca(v) 2.2 and Ca(v) 2.3 channels, and that somatotrophs and lactotrophs share similar proportions of these non-L-type Ca(2+) channels. Furthermore, experiments on mixed populations of native anterior pituitary cells revealed that the fraction of HVA Ca(2+) influx carried through these non-L-type Ca(2+) channels might even be higher (approximately 46%), suggesting that non-L-type channels exist in the majority of native anterior pituitary cells. Using western blotting, immunoblots for α(1C) , α(1D) , α(1A) , α(1B) and α(1E) Ca(2+) channel subunits were identified in native rat anterior pituitary cells. Additionally, using reverse transcriptase-polymerase chain reaction, cDNA transcripts for α(1C) , α(1D) , α(1A) and α(1B) Ca(2+) channel subunits were identified. Transcripts for α(1E) were nonspecific and transcripts for α(1S) were not detected at all (control). Taken together, these results clearly demonstrate the existence of multiple HVA Ca(2+) channels in the membrane of rat native anterior pituitary cells. Whether these channels are segregated among different membrane compartments was investigated further in flotation assays, demonstrating that Ca(v) 2.1, Ca(v) 1.2 and caveolin-1 were mostly localised in light fractions of Nycodenz gradients (i.e. in lipid raft domains). Ca(v) 1.3 channels were distributed among both light and heavy fractions of the gradients (i.e. among raft and nonraft domains), whereas Ca(v) 2.2 and Ca(v) 2.3 channels were distributed mostly among nonraft domains. In summary, in the present study, we demonstrate multiple pathways for HVA Ca(2+) influx through L-type and non-L-type Ca(2+) channels in the membrane of native anterior pituitary cells. The compartmentalisation of these channels among raft and nonraft membrane domains might be essential for their proper regulation by separate receptors and signalling pathways.

MultipleCa2+Channel‐Dependent Components in Growth Hormone Secretion from Rat Anterior Pituitary Somatotrophs

“…We recently identified multiple Ca 2+ channel α1 subunits in the membranes of AP cells, including L‐type (Ca v 1.2, Ca v 1.3), P/Q‐type (Ca v 2.1), N‐type (Ca v 2.2) and R‐type (Ca v 2.3) channels. We also demonstrated that a substantial fraction (approximately 46%) of the VGCI in AP cells is carried through non‐L‐type Ca 2+ channels (P/Q‐, N‐ and R‐type channels) . Additionally, via flotation experiments, we demonstrated that these multiple pathways for VGCI in AP cells are segregated among raft and nonraft membrane compartments .…”

“…We also demonstrated that a substantial fraction (approximately 46%) of the VGCI in AP cells is carried through non‐L‐type Ca 2+ channels (P/Q‐, N‐ and R‐type channels) . Additionally, via flotation experiments, we demonstrated that these multiple pathways for VGCI in AP cells are segregated among raft and nonraft membrane compartments . In the present study, we examined the relative contribution of L‐type and non‐L‐type VGCI to GH secretion.…”

“…Therefore, it is reasonable to assume that ‘stimulated’ GH secretion in our experiments was monitored at membrane potentials near or below 0 mV. Depolarisations to these membrane potentials are expected to produce a maximal VGCI based on our previous whole‐cell experiments in AP cells .…”

“…At higher concentrations, SNX‐482 may block L‐type , N‐type and P/Q‐type currents. Importantly, the same blockers at the same concentrations have been previously used to block high‐voltage activated (HVA) Ca 2+ channel currents (with Ba 2+ as a charge carrier) in AP cells . Nifedipine was purchased from Sigma and the peptide toxins were obtained from Alomone Labs (Jerusalem, Israel).…”

“…Detergent‐resistant membranes (DRMs) were isolated from extracted membranes (see membrane extraction) using Nycodenz gradients as described previously . Briefly, 500 μl of extracted membranes were added to 50 μl of ice‐cold Triton buffer (25 m m Tris HCl, pH 7.5, 150 m m NaCl, 5 m m EDTA and 10% Triton ×100) for 15 min at an ice‐cold temperature (the final concentration of Triton was approximately 1%).…”