M. Sanchezfernandez et al., MOBILIZATION OF INTRACELLULAR CALCIUM IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS BY URIDINE TRIPHOSPHATE AND THE CALCIUM IONOPHORE A23187, The Journal of membrane biology, 135(3), 1993, pp. 273-287
The known action of uridine triphosphate (UTP) to contract some types
of vascular smooth muscle, and the present finding that it is more pot
ent than adenosine triphosphate in eliciting an increase in cytosolic
Ca2+ concentration in aortic smooth muscle, led us to investigate the
mode of action of this nucleotide. With this aim, cultured bovine aort
a cells were subjected to patch-clamp methodologies under various cond
itions. Nucleotide-induced variations in cytosolic Ca2+ were monitored
by using single channel recordings of the high conductance Ca2+-activ
ated K+ (Maxi-K) channel within on-cell patches as a reporter, and who
le-cell currents were measured following perforation of the patch. In
cells bathed in Na+-saline, UTP (>30 nM) induced an inward current, an
d both Maxi-K channel activity and unitary current amplitude of the Ma
xi-K channel transiently increased. Repetitive exposures elicited simi
lar responses when 5 to 10 min wash intervals were allowed between cha
llenges of nucleotide. Oscillations in channel activity, but not oscil
lation in current amplitude were frequently observed with UTP levels >
0.1 muM. Cells bathed in K+ saline (150 mM) were less sensitive to UT
P (approximately 5-fold), and did not show an increase in unitary Maxi
-K current amplitude. Since the increase in amplitude occurs due to de
polarization of the cell membrane, a change in amplitude was not obser
ved in cells previously depolarized with K+ saline. The enhancement of
Maxi-K channel activity in the presence of UTP was not diminished by
Ca2+ entry blockers or by removal of extracellular Ca2+. However, in t
he latter case, repetitive responses progressively declined. These obs
ervations, as well as data comparing the action of low concentrations
of Ca2+ ionophores (<5 muM) to that of UTP indicate that both agents e
levate cytosolic Ca2+ by mobilization of this ion from intracellular p
ools. However, the Ca2+ ionophore did not cause membrane depolarizatio
n, and thus did not change unitary current amplitude. The effect of UT
P on Maxi-K channel activity and current amplitude was blocked by pert
ussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not
modified by okadaic acid, or by inhibitors of protein kinase C (PKC).
Our data support a model in which a pyrimidinergic receptor is coupled
to a G protein, and this interaction mediates release of Ca2+ from in
tracellular pools, presumably via the phosphatidyl inositol pathway. T
his also results in activation of membrane channels that give rise to
an inward current and depolarization. Ultimately, smooth muscle contra
ction ensues. PKC does not appear to be directly involved, even though
the UTP response is blocked by low nm levels of PMA. While the latter
data implicate PKC in diminishing the UTP response, agents that inhib
it either PKC or phosphatase activity did not prevent abolition of UTP
responses by PMA, nor did they modify basal channel activity.