L‐, N‐ and T‐ but neither P‐ nor Q‐type Ca 2+ Channels Control Vasopressin‐Induced Ca 2+ Influx in Magnocellular Vasopressin Neurones Isolated from the Rat Supraoptic Nucleus

1 The role of voltage‐dependent Ca 2+ channels during vasopressin and oxytocin actions on their respective neurones has been analysed by measuring intracellular Ca 2+ concentration ([Ca 2+ ] i ) in individual, freshly dissociated magnocellular neurones from rat supraoptic nucleus (SO) using microspectrofluorimetry. 2 Pre‐incubation of vasopressin‐sensitive neurones with Cd 2+ (100μM), a non‐discriminatory high‐voltage‐activated Ca 2+ channel antagonist, or Ni 2+ (50μM), a blocker of T‐type Ca 2+ current, reduced [Ca 2+ ] i responses by 77 and 19%, respectively. When Cd 2+ was given together with Ni 2+ , the response was blocked by 92%. Similarly, when Ni 2+ was pre‐incubated with Cd 2+ , the response was blocked by ∼84%. 3 Exposure of vasopressin‐sensitive neurones to a specific Ca 2+ channel blocker, nicardipine (L‐type) reduced vasopressin responses by 48% at 1μM and 62% at 5μM. Similarly, ω‐conotoxin GVIA (ω‐CgTX, N‐type; 500 n m ) inhibited the response by 46% with a stronger inhibition (75%) at 800 n m . By contrast, neither ω‐agatoxin IVA (ω‐Aga IVA; 300 n m ), which blocks both P‐ and Q‐type channels, nor synthetic ω‐conotoxin MVIIC (ω‐MVIIC; 100 or 500 n m ), a Q‐type blocker, affected vasopressin‐induced [Ca 2+ ] i responses. These antagonists, given together (nicardipine 5μM +ω‐CgTX 800 n m +ω‐Aga IVA 300 n m ), decreased vasopressin‐induced [Ca 2+ ] i responses by 76 %. 4 In vasopressin‐sensitive neurones, the presence of both nicardipine and ω‐CgTX, reduced the K + ‐evoked [Ca 2+ ] i increase by 61 %. This blockade was increased by a further 21 % with ω‐Aga IVA, suggesting that N‐, L‐ and P‐type channels contribute to the depolarization‐induced [Ca 2+ ] i rise. In addition, ω‐MVIIC alone reduced the K + ‐evoked [Ca 2+ ] i release by 24%. Also the remaining K + responses were further reduced by 60% when pre‐incubated with L‐ N‐ and P‐type blockers, suggesting the involvement of Q‐type channels. 5 In oxytocin‐sensitive neurones, the peak amplitude of the [Ca 2+ ] i response was not affected by Cd 2+ alone, by combined Cd 2+ and Ni 2+ , or by the mixture of nicardipine, ω‐CgTX and ω‐Aga IVA. By contrast, the responses evoked by depolarization with K + were blocked by Cd 2+ . Both nicardipine and ω‐CgTX blocked 65% of K + response and an additional block of ∼18% was obtained with ω‐Aga IVA, suggesting the involvement of L‐, N‐ and P‐type channels. In combination, these antagonists strongly inhibited (∼80% reduction) the K + responses. Further reduction to 18% was made by the Q‐type blocker ω‐MVIIC. Pre‐incubation with L‐, N‐ and P‐type blockers caused an additional block of 71 %. 6 Some supraoptic neurones (5–10%) responded to both vasopressin and oxytocin, with only the [Ca 2+ ] i responses induced by vasopressin blocked (> 90% inhibition) by the mixture of Ca 2+ channel antagonists. 7 In conclusion, both vasopressin and oxytocin magnocellular SO neurones have been shown to express T‐, L‐, N‐, P‐, Q‐ and R‐type Ca 2+ channels in their somata. Our results show that the vasopressin‐induced [Ca 2+ ] i increase in vasopressin‐sensitive neurones is mediated by L‐, N‐and T‐type Ca 2+ channels and not by P‐ and Q‐type channels; Ca 2+ channels are not involved in oxytocin action on oxytocin‐sensitive neurones and L‐, N‐, P‐ and Q‐type channels control the K + ‐induced [Ca 2+ ] i increase in SO neurones.