SUMMARY1. K-sensitive electrodes placed in the extracellular fluid have been used to show that ATP and noradrenaline cause a rapid loss of up to 10 % of the K content of isolated guinea-pig hepatocytes.2. The hypothesis that this response is a consequence of a rise in the K permeability of the hepatocyte membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated K loss, in this instance of up to 20-25 % of the amount in the cells.3. Under similar conditions A23187 caused a transient increase, followed by a larger decrease, in the 45Ca content of guinea-pig hepatocytes equilibrated with this isotope. The decrease alone was seen with ATP and noradrenaline.4. Quinine (1 mM) and the bee venom neurotoxin apamin (10 nM) greatly reduced the effect of ATP, noradrenaline and A23187 on K content without affecting the changes in 45Ca movement.5. Apamin (10 nM) also abolished the increase in 42K efflux which follows the application of the a-adrenoceptor agonist amidephrine to rabbit liver slices; the concurrent rises in 45Ca efflux and glucose release were unaffected.6. It was concluded that quinine and apamin are able to block either the Ca-dependent K channels present in guinea-pig and rabbit liver cell membranes or the mechanism that controls them.7. Surprisingly, rat hepatocytes took up rather than lost K when treated with the concentrations of ATP, noradrenaline or A23187 that initiated K loss from guinea-pig cells. This response was greatly reduced by ouabain.8. Application of large concentrations of A23187 to rat hepatocytes caused K loss associated with cell death.9. The influence of apamin (10-1000 nM) and quinine (200-1000 UM) on the Ca-dependent K permeability of red blood cells and ghosts was also studied. Apamin was without effect even when applied to both sides of the ghost membrane, whereas quinine caused inhibition, as reported by others.10. The results suggest that Ca-dependent K channels or carriers are present in

Apamin is a neurotoxic polypeptide of known structure isolated from bee venom. Shuba and coworkers have recently shown that it abolishes the hyperpolarising action of externally-applied ATP on visceral smooth muscle (guinea pig stomach and taenia coli) as well as the hyperpolarisation (inhibitory junction potential) that follows stimulation of the non-adrenergic inhibitory nerve supply to these tissues. As it has been proposed that ATP is the neurotransmitter involved in the latter response, Vladimirova and Shuba tentatively concluded that apamin is a specific postsynaptic blocking agent of this non-adrenergic, possibly 'purinergic', inhibition. We have confirmed the important observation that nanomolar concentrations of apamin reduce inhibition by ATP and by non-adrenergic nerve stimulation, but further experiments suggest that, rather than acting as a specific blocker of ATP receptors, apamin inhibits the increase in potassium permeability caused by a number of agents, including ATP.

Abstract-Despite intensive interest in the dedifferentiation process of vascular smooth muscle cells, very little data are available on intracellular Ca 2ϩ signaling. The present study was designed to investigate the evolution of the intracellular Ca 2ϩ pools when rat aortic smooth muscle cells (RASMCs) proliferate and to define the mechanisms involved in the functional alterations. RASMCs were cultured in different conditions, and [Ca 2ϩ ] i was measured by use of fura 2. Expression of the sarco(endo)plasmic reticulum Ca 2ϩ pumps (SERCA2a and SERCA2b), Ca 2ϩ channels, the ryanodine receptor (RyR), and the inositol trisphosphate receptor (IP3R) was studied by reverse transcription-polymerase chain reaction and immunofluorescence. Antibodies specific for myosin heavy chain isoforms were used as indicators of the differentiation state of the cell, whereas an anti-proliferating cell nuclear antigen antibody was a marker of proliferation. SERCA2a, SERCA2b, RyR3, and IP3R-1 mainly were present in the aorta in situ and in freshly isolated RASMCs. These cells used the 2 types of Ca 2ϩ channels to release Ca 2ϩ from a common thapsigargin-sensitive store. Proliferation of RASMCs, induced by serum or by platelet-derived growth factor-BB, resulted in the disappearance of RyR and SERCA2a mRNAs and proteins and in the loss of the caffeine-and ryanodine-sensitive pool. The differentiated nonproliferative phenotype was maintained in low serum or in cells cultured at high density. In these conditions, RyR and SERCA2a were also present in RASMCs. Thus, expression of RyR and SERCA2a is repressed by cell proliferation, inducing loss of the corresponding Ca 2ϩ pool. In arterial smooth muscle, Ca 2ϩ release through RyRs is involved in vasodilation, and suppression of the ryanodine-sensitive pool might thus alter the control of vascular tone.