Expression of plasma membrane calcium ATPases in phenotypically distinct canine vascular smooth muscle cells.

Our laboratory has identified at least two types of vascular smooth muscle cells (VSMCs) that exist in canine arteries and veins: type 1 cells, located in the media express muscle specific proteins but do not proliferate in culture; and type 2 cells, located in both media and adventitia, do not express muscle specific protein but proliferate in culture. Plasma membrane Ca(2+)-ATPases (PMCAs) have been implicated in proliferation control. The present study examines the expression of PMCA isoforms and calmodulin-binding domain splice variants in these two types of canine VSMCs. PMCA protein was found in both type 1 and type 2 cells. Reverse transcriptase-polymerase chain reaction assays were developed for canine PMCA calmodulin-binding domain splice variants. We cloned and sequenced isolates corresponding to PMCA1b, 4a and 4b from canine VSMCs. PMCA 2 and 3 were not detected. Freshly isolated type 1 cells expressed PMCA 1b, 4a and 4b, while freshly isolated type 2 cells expressed PMCA1b and 4b. Upon placement in culture, type 2 cells originating from either carotid artery or saphenous vein demonstrated a time-dependent upregulation of PMCA4a mRNA. Treatment with the phosphoinositide 3-kinase inhibitor wortmannin produced concentration-dependent inhibition of both PMCA4a upregulation and [(3)H]thymidine incorporation. These findings suggest a role for phosphoinositide 3-kinase in regulating PMCA expression, which may be important in the control of Ca(2+)-sensitive VSMC functions.

Differentiated vascular myocytes: are they involved in neointimal formation?

The role of differentiated vascular myocytes are neointimal formation in canine carotid artery was investigated. Using antibodies and cDNA probes, cells were characterized in situ and after isolation. In situ characterization indicated the majority of medial cells expressed both smooth muscle myosin and alpha actin but many cells were negative to these markers. All adventitial cells were negative for these proteins. The muscle protein-positive cells were designated differentiated, vascular myocytes (VSMC). The others were designated type 2 cells. Sequential enzyme digestion from lumenal surface yielded VSMC ( > 90%) while digestions from the adventitial surface yielded type 2 cells ( > 90%). VSMC were viable in culture but did not spread, proliferate, or alter expression of muscle proteins. Type 2 cells proliferated and increased their expression of muscle actin but did not express muscle myosin. Characterization of neointimal cells from injured carotid arteries indicated they were morphologically and immunologically identical to cultured type 2 cells. We concluded that: (a) canine carotid artery media consists of a heterogeneous cell population: (b) serum does not stimulate isolated VSMC to undergo phenotypic modulation or proliferate: and (c) type 2 cells may be responsible for neointimal formation because they proliferate and acquire a phenotype identical to in situ neointimal cells.

Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells.

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [3H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.

Sodium pump stimulation by activation of two alpha adrenergic receptor subtypes in canine blood vessels.

The effects of alpha-1 and alpha-2 selective adrenergic agents on sodium pump activity were investigated in intact canine femoral artery and saphenous vein by measuring ouabain-sensitive uptake of 86Rb. In both vessels, the alpha-1-selective agonist, phenylephrine, stimulated 86Rb uptake in a dose-dependent manner. The uptake was blocked by prazosin and yohimbine with the order of potency: prazosin greater than yohimbine. The alpha-2-selective agonist, clonidine, also stimulated 86Rb uptake in the saphenous vein but not in the femoral artery. The stimulation was blocked by prazosin and yohimbine with the order of potency: yohimbine greater than prazosin. The potency of phenylephrine to contract saphenous vein or femoral artery was the same as that for stimulation of ouabain-sensitive 86Rb uptake. Clonidine was 10-fold more potent as a contractile agonist than as a Na+ pump stimulant. It caused only a weak contraction in the femoral artery. Reducing extracellular sodium abolished the stimulation of 86Rb uptake by both phenylephrine and clonidine in saphenous vein. Subsequently it was shown that both agonists increased intracellular sodium levels and these increases were blocked by the alpha receptor antagonists, prazosin and yohimbine, with the same selectivity as was observed in the 86Rb uptake experiments. Sodium pump stimulation produced by both phenylephrine and clonidine was blocked by amiloride. These observations suggest that the activity of the vascular sodium pump can be regulated by both alpha-1 and alpha-2 adrenergic receptors and that the mechanism involves an influx of sodium, most likely through a stimulation of Na+/H+ exchange.