Long lasting inhibition of adenylyl cyclase selectively mediated by inositol 1,4,5-trisphosphate-evoked calcium release.

In A7r5 smooth muscle cells, vasopressin stimulates release of Ca2+ from intracellular stores and Ca2+ entry, and it inhibits adenylyl cyclase (AC) activity. Inhibition of AC is prevented by inhibition of phospholipase C or when the increase in cytosolic [Ca2+] is prevented by the Ca2+ buffer, BAPTA. It is unaffected by pertussis toxin, inhibition of protein kinase C, or L-type Ca2+ channels or by removal of extracellular Ca2+. The independence of extracellular Ca2+ occurs despite inhibition of AC by vasopressin persisting for at least 15 min, whereas the cytosolic [Ca2+] returns to its basal level within 1-2 min in Ca2+-free medium. Although capacitative Ca2+ entry (CCE), activated by emptying stores with thapsigargin, inhibits AC, Ca2+ entry via CCE or L-type Ca2+ channels activated by vasopressin is ineffective. Temporally separating vasopressin-evoked Ca2+ release from the assessment of AC activity revealed that the transient Ca2+ signal resulting from Ca2+ mobilization causes a long lasting inhibition of AC. By contrast, inhibition of AC by thapsigargin-evoked CCE reverses rapidly after removal of extracellular Ca2+. Inhibition of AC by vasopressin is prevented by inhibition of Ca2+-calmodulin-dependent protein kinase II. We conclude that persistent inhibition of AC (probably AC-3) by vasopressin is mediated by inositol trisphosphate-evoked Ca2+ release causing activation of Ca2+-calmodulin-dependent protein kinase II. Our results establish that an important interaction between two ubiquitous signaling pathways is tuned selectively to Ca2+ release via inositol trisphosphate receptors and that the interaction transduces a transient Ca2+ signal into a long lasting inhibition of AC.

Differential effect of PKA on the Ca2+ release kinetics of the type I and III InsP3 receptors.

The effects of protein kinase A (PKA) on the inositol 1,4,5-trisphosphate (InsP(3)) receptor isoforms type I and type III were studied. The effects of PKA on the extent and rate constants for InsP(3)-induced Ca(2+) release (IICR) were different for the two isoforms. The effects of PKA on the type I isoform showed a biphasic relationship dependent upon the concentration of PKA used. At low concentrations of PKA (<50U/ml), both the extent and rate constants for IICR increased, while at higher concentrations (>200U/ml) the extent and rate constants decreased. The type III isoform showed only an increase in the extent of IICR and not in the rate constants. The effects of PKA on the type I InsP(3) receptor using single channel electrophysiological studies were also investigated. The stimulatory effect of PKA is due to an increase in conductance levels and not to a change in the mean open time of the channel.

Nitric oxide co-ordinates the activities of the capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin.

In A7r5 vascular smooth muscle cells vasopressin, via arachidonic acid, regulates two Ca(2+)-entry pathways. Capacitative Ca(2+) entry (CCE), activated by empty Ca(2+) stores, is inhibited by arachidonic acid, and non-capacitative Ca(2+) entry (NCCE) is stimulated by it. This reciprocal regulation ensures that all Ca(2+) entry is via NCCE in the presence of vasopressin, while CCE mediates a transient Ca(2+) entry only after removal of vasopressin. We demonstrate that type III NO synthase (NOS III) is expressed in A7r5 cells and that NO inhibits CCE. Inhibition of CCE by vasopressin requires NOS III and the requirement lies downstream of arachidonic acid. Activation of soluble guanylate cyclase by NO and subsequent activation of protein kinase G are required for inhibition of CCE. Stimulation of NCCE by vasopressin also requires NOS III, but the stimulation is neither mimicked by cGMP nor blocked by inhibitors of soluble guanylate cyclase or protein kinase G. We conclude that arachidonic acid formed in response to vasopressin stimulates NOS III. NO then directly stimulates Ca(2+) entry through NCCE and, via protein kinase G, it inhibits CCE. The additional amplification provided by the involvement of guanylate cyclase and protein kinase G ensures that CCE will always be inhibited when vasopressin activates NCCE.