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.

The involvement of L-type Ca(2+) channels in both 'basal' and 'stimulated' growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non-L-type Ca(2+) channels is lacking. We investigated whether non-L-type Ca(2+) channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15 min with 2 mm K(+) ('basal' secretion) or 60 mm K(+) ('stimulated' secretion). The role of non-L-type Ca(2+) influx was investigated using specific channel blockers, including ω-agatoxin-IVA, ω-conotoxin GVIA or SNX-482, to block P/Q-, N- or R-type Ca(2+) channels, respectively. Our results demonstrate that P/Q-, N- and R-type Ca(2+) channels contributed 21.2 ± 1.9%, 20.2 ± 7.6% and 11.4 ± 1.8%, respectively, to 'basal' GH secretion and 18.3 ± 1.0%, 24.4 ± 5.4% and 14.2 ± 4.8%, respectively, to 'stimulated' GH secretion. After treatment with a 'cocktail' that comprised the previously described non-L-type blockers, non-L-type Ca(2+) channels contributed 50.9 ± 0.4% and 45.5 ± 2.0% to 'basal' and 'stimulated' GH secretion, respectively. Similarly, based on the effects of nifedipine (10 μM), L-type Ca(2+) channels contributed 34.2 ± 3.7% and 54.7 ± 4.1% to 'basal' and 'stimulated' GH secretion, respectively. Interestingly, the relative contributions of L-type/non-L-type Ca(2+) channels to 'stimulated' GH secretion were well correlated with the relative contributions of L-type/non-L-type Ca(2+) channels to voltage-gated Ca(2+) influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca(2+) channels is important for GH secretion. Lipid raft disruption (methyl-β-cyclodextrin, 10 mm) abrogated the compartmentalisation of Ca(2+) channels and substantially reduced 'basal' and 'stimulated' GH secretion by 43.2 ± 3.4% and 58.4 ± 4.0%, respectively. In summary, we have demonstrated that multiple Ca(2+) channel-dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.