Opioid peptide modulation of Ca(2+)-dependent K+ and voltage-activated Ca2+ currents in bovine adrenal chromaffin cells.

Opioid peptides are abundantly expressed in the adrenal medulla, and there is evidence that they may be released presynaptically or as medullary paracrine agents. To assess the physiological relevance of these observations, we investigated opioid effects on ionic currents from cultured bovine adrenal chromaffin cells. Under whole-cell path-clamp conditions, opioid peptides, acting via a mu-type opioid receptor, strongly potentiated the large conductance Ca(2+)-dependent K+ (BK) channel current. Opioids also inhibited voltage-activated Ca2+ currents. Application of opioid peptides to the extracellular face of outside-out patches also increased opening activity of single BK channels, suggestive of tight receptor-channel coupling. This potentiating effect on BK current, combined with the inhibition of Ca2+ current, indicates that opioids may have an inhibitory influence on secretory activity of the adrenal medulla. The widespread distribution of the BK channel class suggests that the significance of its modulation by opioids could also extend beyond the adrenal gland.

Nucleotide-independent modulation of Ca(2+)-dependent K+ channel current by a mu-type opioid receptor.

Physiological responses to opiates and opioid peptides are transduced via receptors coupled to G proteins. The effectors for these G proteins are often ion channels or second messenger systems that modulate channel activity. In cultured bovine adrenal medullary chromaffin cells (BAMCCs), the activity of a calcium-dependent, voltage-sensitive, potassium (BK) channel is robustly potentiated by a mu-type opioid receptor, an effect consistent with the inhibitory role of opioids versus neural excitability. Patch-clamp electrophysiology was used to investigate coupling between the mu receptor and BK channel, leading to rather surprising results. Potentiation of BK channel activity by the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10 nM) was unaffected by all attempts to disrupt or alter G protein function, including incubation of cells with pertussis toxin (PTX) and inclusion of guanosine 5'-O-(2-thio)diphosphate (GDP beta S) or guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) in intracellular recording solutions. However, dopamine D2 receptor potentiation of BK current in these same cells was affected by PTX, GDP beta S, and GTP gamma S in predictable fashion. Thus, PTX and GDP beta S inhibited dopamine potentiation of BK current, and GTP gamma S prolonged reversal of dopamine action. These results suggest that the BAMCC BK channel is not coupled to the mu receptor via a GTP-dependent mechanism, whereas in the same cells the dopamine D2 receptor modulates BK channel activity in a conventional GTP-dependent manner. In addition, replacement of both ATP and GTP with nonhydrolyzable analogs also failed to affect either potentiation or recovery of BK channel activity in response to [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin. These results indicate that in BAMCCs the mu-opioid receptor modulates BK channel activity independently of either G proteins or phosphorylation-dependent processes.

Ca2+-dependent K+ channels in bovine adrenal chromaffin cells are modulated by lipoxygenase metabolites of arachidonic acid.

Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to potentiate activity of the Ca2+ and voltage-dependent, large-conductance K+ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca2+ buffering ruled out cytoplasmic messengers or changes in intracellular Ca2+ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting action potential duration and thus voltage-gated Ca2+ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs.