INDEPENDENT EXTERNAL CALCIUM-ENTRY AND CELLULAR CALCIUM MOBILIZATION IN XENOPUS-OOCYTES

We studied cellular calcium (Ca) mobilization and Ca entry from the me dium following injection of various inositol phosphates (IPs) or activ ation of thyrotropin-releasing hormone receptors (TRH-Rs) in oocytes i njected with TRH-R cRNA. We determined the order of potency of various IPs for evoking the rapid depolarizing current in Ca-free medium, whi ch reflects the mobilization of cellular Ca. The most potent compound was inositol 1,4,5-trisphosphate (lns(1,4,5)P-3), followed by inositol 1,2,4,5-tetrakisphosphate (lns(1,2,4,5)P-4), which displayed 91% of t he activity of lns(1,4,5)P-3, while inositol 1,3,4,6-tetrakisphospkate (lns(1,3,4,6)P-4) had only 29% effect. All other IPs used in the pres ent study exhibited responses that were 40% or less than those elicite d by lns(1,4,5)P-3. Cellular Ca mobilization was confirmed by Ca-45(2) efflux for lns(1,4,5)P-3, lns(1,2,4,5)P-4 and lns(1,3,4,6)P-4, or by Fura-2 ratio imaging studies for the latter. In parallel, we assayed the ability of these compounds to promote Ca entry into the cell, as r eflected by Ca-evoked depolarizing current or Fura-2 imaging. These as says revealed a different order of potency, where lns(1,4,5)P-3 > inos itol 4,5-bisphosphate (lns(4,5)P-2) > lns(1,3,4,6)P-4 = lns(1,2,4,5)P- 4. All other inositol phosphates were largely ineffective. Heparin inh ibited the response to TRH by 67% while Ca entry was inhibited only by 22%. The latency of the response to TRH was significantly shorter in the presence of extracellular Ca, suggesting Ca entry preceded the res ponse, i.e. major depletion of Ca stores. These results strongly sugge st that the activation of Ca entry is largely independent of cellular Ca mobilization and may be mediated by a receptor for an unidentified phosphorylated compound, different from that for lns(1,4,5)P-3 on the endoplasmic reticulum.