Low concentrations of inositol 1,4,5-trisphosphate (InsP3) evoke a very rapid mobilization of intracellular Ca2+ stores in many cell types, which can be followed by a further, much slower efflux. Two explanations have been suggested for this biphasic release. The first proposes that the Ca2+ stores vary in their sensitivity to InsP3, and each store releases either its entire contents or nothing (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP3, but that they can release only a fraction of their Ca2+ before their sensitivity is somehow attenuated (steady-state release). Experiments using purified InsP3 receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP3 under conditions in which the total Ca2+ level at both sides of the receptor is held constant. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca(2+)-free medium, the amount of 45Ca2+ remaining in the stores after the rapid transient phase of release is independent of their initial Ca2+ levels, indicating that partially depleted stores are less sensitive to InsP3. Moreover, if the stores are reloaded with 40Ca2+ after the first stimulus, reapplication of the same low concentration of InsP3 will release further 45Ca2+. This recovery of InsP3 sensitivity is almost complete. Under these conditions, Ca2+ release must thus occur by a steady-state mechanism, in which the decreasing Ca2+ content of the stores slows down further release.

3. At the same [Ca]. the degree of filling is higher in K-depolarized tissues than in control tissues. However at 10 mM- [Ca]. and 5-9 mM-K the amount of Ca taken up by the store is larger than that after loading in 0-2 mM-Ca and 141-4 mM-K, although the tissues remain relaxed during loading at 5 9 mM-K and contracted at 141-4 mM-K.4. The Ca antagonists D600 and nicardipine selectively block the contraction induced by K depolarization, but do not affect appreciably the noradrenaline-induced contraction.5. The filling of the store is not significantly reduced by the presence of the Ca antagonists in solutions containing 5-9 mM-K. However these antagonists reduce the degree of filling in K-rich loading solution to a level which is lower than that observed in the control.6. Mn blocks both the contraction induced by K-rich solution and the tonic component of the noradrenaline-induced contraction and it also inhibits filling of the store.7. The results suggest that the filling of the store under physiological conditions occurs by a direct pathway between the store and the extracellular medium.

A ~ S T R A C T A contraction of the rabbit ear artery can be induced by depolarizing the cells with a K-rich solution if Ca is present. 10 -9-10 -8 M noradrenaline and 10 -s-10 -7 M histamine cause a contraction of this tissue without modifying the membrane potential. If the histamine concentration exceeds 10 -~ M some depolarization of the membrane also occurs. Both noradrenaline and histamine also induce a contraction in Ca-free medium, even if La is present. None of these stimuli produces action potentials or fluctuations of the membrane potential. Besides these tonic contractions, the ear artery can also produce phasic contractions when 10 mM TEA is added to the medium. Such contractions are caused by the appearance of action potentials which are Ca dependent and which are similar to those appearing in visceral smooth muscle. A study of 4SCa fluxes has revealed that K depolarization and noradrenaline cause only a small increase in 4SCa uptake by the cells, while noradrenaline also releases cellular Ca, even in Ca-free medium. A comparison of tension development and 45Ca release induced by noradrenaline in Ca-free medium suggests that Ca extrusion could be very efficient in the rabbit ear artery and that it could play a direct role in its relaxation.

SUMMARY1. The membrane potential of the smooth muscle cells of-the rabbit main pulmonary artery amounts to -57 mV, the length constant of the tissue is 1-48 mm and the time constant of the membrane 182 msec. On the basis of the electrical properties of its membrane, this smooth muscle tissue is classified as a single-unit type. During outward currentpulses, the membrane shows marked rectification and action potentials can never be generated.2. Tetraethylammonium (10 mM) and procaine (5 mM) depolarize the membrane and increase the membrane resistance. By studying the effect of both substances on the 42K efflux, it could be concluded that they reduce the K-permeability of the membrane. They also suppress the rectification of the membrane and increase the length constant of the membrane. In the presence of TEA and procaine, a graded response of the membrane can be induced by outward current pulses, but overshoot potentials never occur.3. Noradrenaline, in concentrations between 2 x 10-and 10-7 m, evokes contraction without depolarizing the membrane. When the concentration is increased above 2 x 10-7 M, noradrenaline depolarizes the membrane and reduces the membrane resistance. A study of the effect of noradrenaline on the K, Cl and Na fluxes has revealed that it increases the permeability of the membrane for these three ions.4. The tissue concentrations of Na and K are 80 and 38 m-mole/kg wet wt., respectively.

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