Protein kinase C mediates insulin-inhibited Ca2+ transport and contraction of vascular smooth muscle.

Insulin acutely inhibits contraction of primary cultured vascular smooth muscle (VSM) cells from canine femoral artery by inhibiting contractile agonist-induced Ca2+ influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca2+ concentration (Ca2+i) by stimulating cyclic guanosine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC). Ca2+ influx was assessed by measuring the rate of fluorescence quenching of intracellular fura 2 by extracellular Mn2+. We found that 10 micromol/L serotonin (5-HT) stimulated Mn2+ influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT-stimulated component of Mn2+ influx by 63% (P < .05), but insulin had no effect in the presence of 1 micromol/L staurosporine, an inhibitor of PKC. In the absence of insulin, preincubating cells with 0.1 micromol/L phorbol 12-myristate 13-acetate (PMA) for 5 min inhibited the 5-HT-stimulated component of Mn2+ influx by 69% (P < .05). Insulin inhibited cell contraction induced by raising Ca2+i to supraphysiologic levels with ionomycin by 75% (P < .05). We also noted that 10(-6) mol/L calphostin C, another PKC inhibitor, or 16-h preincubation with PMA completely blocked this effect of insulin. Finally, 10-min exposure to insulin or PMA increased cyclic GMP production in ionomycin-treated cells by 50% and 64%, respectively (both P < .05). We conclude that insulin inhibits VSM cell contraction by inhibiting 5-HT-stimulated Ca2+ influx and also at step(s) distal to Ca2+i by a PKC-dependent mechanism.

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.

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential.

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.

Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase.

Vascular smooth muscle cell (VSMC) migration participates in atherosclerosis and arterial restenosis after balloon angioplasty. Because these processes are enhanced in insulin-resistant states, our goal was to determine whether insulin affects VSMC migration and, if so, how. The migration of primary cultured VSMCs from canine femoral artery was measured with the use of a wound migration assay and related to cGMP levels. Insulin (1 nmol/L) did not affect migration or cGMP production in control cells. When inducible nitric oxide synthase (iNOS) was induced by 24-hour preincubation with lipopolysaccharide and interleuken-1beta, basal migration decreased, cGMP production increased, and insulin inhibited migration by >90% and stimulated cGMP production by 3-fold. The nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine blocked the affect of insulin on the migration of VSMCs with iNOS. 8-Bromo-cGMP inhibited VSMC migration in control cells, and 1-H-1[1,2,4]oxadiazolo-[4, 3a]quinoxolin-1-one, a selective inhibitor of guanylate cyclase, blocked the inhibition by insulin of migration of cells with iNOS. We conclude that insulin does not normally affect cGMP production or the migration of these VSMCs. However, after the induction of iNOS, insulin stimulates cGMP production and inhibits migration via an NOS-and a cGMP-dependent mechanism.

Insulin inhibits vascular smooth muscle contraction at a site distal to intracellular Ca2+ concentration.

Several hypertensive states are associated with resistance to insulin-induced glucose disposal and insulin-induced vasodilation. Insulin can inhibit vascular smooth muscle (VSM) contraction at the level of the VSM cell, and resistance to insulin's inhibition of VSM cell contraction may be of pathophysiological importance. To understand the VSM cellular mechanisms by which insulin resistance leads to increased VSM contraction, we sought to determine how insulin inhibits contraction of normal VSM. It has been shown that insulin lowers the contractile agonist-stimulated intracellular Ca2+ (Ca2+i) transient in VSM cells. In this study, our goal was to see whether insulin inhibits VSM cell contraction at steps distal to Ca2+i and, if so, to determine whether the mechanism is dependent on nitric oxide synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral artery were bathed in a physiological concentration of extracellular Ca2+ and permeabilized to Ca2+ with a Ca2+ ionophore, either ionomycin or A-23187. The resultant increase in Ca2+i contracted individual cells, as measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca2+i or the ionomycin-induced increase in Ca2+i, as determined by fura 2 fluorescence measurements, but it did inhibit ionomycin- and A-23187-induced contractions by 47 and 51%, respectively (both P < 0.05). In the presence of 1.0 microM ionized Ca2+, ionomycin-induced contractions were inhibited by insulin in a dose-dependent manner. In the presence of ionomycin, insulin increased cGMP production by 43% (P < 0.05). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), a selective inhibitor of guanylate cyclase that blocked cGMP production in these cells, completely blocked the inhibition by insulin of ionomycin-induced contraction. It was found that the cells expressed the inducible isoform of NOS. NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester (0.1 mM), inhibitors of NOS, did not affect ionomycin-induced contraction but prevented insulin from inhibiting contraction. We conclude that insulin stimulates cGMP production and inhibits VSM contraction in the presence of elevated Ca2+i. This inhibition by insulin of VSM contraction at sites where Ca2+i could not be rate limiting is dependent on NOS and cGMP.

Insulin acutely inhibits cultured vascular smooth muscle cell contraction by a nitric oxide synthase-dependent pathway.

Insulin acutely decreases contractile agonist-induced Ca2+ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca2+ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase-dependent pathway. Primary cultures of endothelial cell-free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10(-5) mol/L serotonin insulin stimulated cGMP production by 60%. N(G)-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)-stimulated Mn+2 (a Ca2+ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT-stimulated Ca2+ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase-dependent mechanism.

Migratory abilities of different vascular cells from the tunica media of canine vessels.

In response to injury, vascular smooth muscle cells (VSMC) are thought to migrate toward the area of damage, where they participate in the reparative process. We have recently identified and isolated two distinct cell types (VSMC and type 2 cells) from the tunica media of canine carotid artery and saphenous vein. The purpose of these experiments was to determine whether both cell types were able to migrate in response to a variety of chemoattractants. A multiwell Boyden chamber and a wound migration assay were used to assess the migratory ability of these cells in vitro. The results indicated that VSMC did not exhibit directed migration in response to either 10% fetal bovine serum or platelet-derived growth factor (PDGF)-AB. In contrast, type 2 cells migrated to serum, PDGF-AB, and PDGF-BB but not to PDGF-AA, endothelin (ET)-1, or ET-3. No difference in migratory ability was detected between type 2 cells isolated from carotid arteries or saphenous veins. It is concluded that the migratory ability of cells within the tunica media of vessels from adult animals are not equal, suggesting that only selected cells may participate in vascular wall repair.

The A10 cell line: a model for neonatal, neointimal, or differentiated vascular smooth muscle cells?

OBJECTIVES: The A10 cell line was derived from the thoracic aorta of embryonic rat and is a commonly used model of vascular smooth muscle cells (VSMC). Despite its wide use this cell line has not been well characterized. This is especially important in light of recent evidence of phenotypically distinct cell populations isolated from rat vascular tissue. Therefore, the present study was undertaken to confirm the VSMC nature of A10 cells and to investigate whether these cells particularly resemble adult, neonatal, or neointimal rat VSMC. METHODS: A variety of defining characteristics were used that included immunofluorescent analysis for smooth muscle alpha-actin, smooth and non-muscle myosin heavy chains, desmin and vimentin; Western analysis for smooth muscle and non-muscle myosin heavy chains; mRNA analysis for smooth muscle myosin heavy chain, calponin, SM22 alpha, tropoelastin and PDGF-B peptide; and functional assays of cell migration, proliferation and agonist induced intracellular Ca transients. RESULTS: A10 cells expressed smooth muscle alpha-actin, SM22 alpha, smooth muscle calponin and vimentin, characteristic of in vivo rat VSMCs; however they also resembled de-differentiated smooth muscle cells in that they expressed non-muscle myosin rather than smooth muscle myosin heavy chain. A10 cells resembled cultured rat neonatal smooth muscle cells ("pup cells") in that they had an epithelioid shape and lacked functional PDGF-alpha receptors: however they did not express PDGF-B mRNA or proliferate in low serum containing medium as do neonatal cells. A10 cells had several characteristics in common with neointimal cells including the expression of alpha-actin, vimentin, and non-muscle myosin and the lack of expression of PDGF-B mRNA as well as the ability to migrate in response to PDGF-BB. CONCLUSION: In conclusion, A10 cells are nondifferentiated VSMC that differ from neonatal but bear significant resemblance to neointimal cells.

Insulin-stimulated glucose transport inhibits Ca2+ influx and contraction in vascular smooth muscle.

BACKGROUND: Insulin attenuates serotonin-induced Ca2+ influx, the intracellular Ca2+ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca2+ influx, intracellular Ca2+ concentration, and contraction in these cells. METHODS AND RESULTS: Insulin 1 nmol/L stimulated the 30-minute uptake of [3H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca2+ concentration was monitored by measuring fura 2 fluorescence by use of Ca(2+)-sensitive excitation wavelengths, and Ca2+ influx was estimated by the rate of Mn2+ quenching of intracellular fura 2 fluorescence when excited at a Ca(2+)-insensitive wave-length. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10(-5) mol/L serotonin-induced contraction of individual cells by 62% (P < .01) and decreased the serotonin-stimulated component of Mn2+ influx by 78% (P < .05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca2+ peak by 37% (P < .05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn2+ influx, the serotonin-induced Ca2+ peak was decreased by 46% (P < .05), serotonin-induced contraction was inhibited by 60% (P < .01), and addition of insulin did not further inhibit contraction. CONCLUSIONS: Since the effects of insulin on serotonin-stimulated Ca2+ transport, intracellular Ca2+ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca2+ influx and contraction in vascular smooth muscle.

Insulin inhibits serotonin-induced Ca2+ influx in vascular smooth muscle.

BACKGROUND: Insulin in physiological concentrations attenuates the agonist-induced intracellular Ca2+ ([Ca2+]i) transient and inhibits contraction in individual nonproliferated cultured canine femoral artery vascular smooth muscle cells (VSMCs). In the present study, we wished to define the effects of insulin on individual components of Ca2+ transport in vascular smooth muscle. METHODS AND RESULTS: Insulin (40 microU/mL) attenuated the 5-hydroxytryptamine (5-HT, serotonin; 10(-5) mol/L)-induced [Ca2+]i transient (measured by fura 2 fluorescence) in primary confluent canine femoral artery VSMCs in the presence of extracellular Ca2+. In Ca(2+)-free media, the 5-HT-induced [Ca2+]i transient was reduced by 42% and was not affected by insulin. This finding suggested that insulin inhibits 5-HT-induced Ca2+ influx but does not affect sarcolemmal Ca2+ efflux or Ca2+ release from intracellular stores. In support of those conclusions, we found that insulin inhibited the 5-HT-induced component of Mn2+ (a Ca2+ surrogate) influx (measured by fura 2 fluorescence quenching at the Ca2+ isosbestic excitation wavelength). In addition, 5-HT stimulated the rates of 45Ca2+ efflux from intact cells (a measure of sarcolemmal Ca2+ efflux) and from saponin-permeabilized cells (a measure of Ca2+ release from intracellular stores), but insulin did not affect these rates of 45Ca2+ efflux. CONCLUSIONS: We conclude that a physiological insulin concentration attenuates the 5-HT-induced [Ca2+]i transient in confluent primary cultured canine femoral artery VSMCs by inhibiting the 5-HT-induced component of Ca2+ influx but not by affecting sarcolemmal Ca2+ efflux or Ca2+ release from intracellular stores.

Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle.

Resistance to insulin-induced glucose disposal is associated with hypertension, in accord with recent reports that insulin-induced vasodilation is impaired in men with resistance to insulin-induced glucose disposal. Nevertheless, the mechanism of insulin-induced vasodilation is not known. We wished to determine whether a physiological concentration of insulin inhibits agonist-induced contraction at the level of the individual vascular smooth muscle cell, and if so, how. Dispersed vascular smooth muscle cells from dog femoral artery were grown on collagen gels for 4 to 8 days. Contraction and intracellular Ca2+ concentration of individual cells were measured by photomicroscopy and fura 2 epifluorescence microscopy, respectively. Serotonin and angiotensin II contracted cells in a dose-dependent manner. Preincubation of cells for 20 minutes (short-term) or 7 days (long-term) with insulin (40 microU/mL) inhibited serotonin- and angiotensin II-induced contractions by approximately 50%. Insulin (10 microU/mL) acutely inhibited serotonin-induced contraction by 34%. The maximal effect of high extracellular K(+)-induced contraction was not affected by short-term insulin exposure, but the ED50 for extracellular K(+)-induced contraction was increased from 7.6 +/- 2.5 to 16.0 +/- 3.9 mmol/L (P < .05). Short-term insulin exposure also attenuated the peak rise of the serotonin-induced intracellular Ca2+ transient and increased the rate constant for intracellular Ca2+ decline. Verapamil and ouabain completely blocked the attenuation of agonist-induced contraction by short-term insulin exposure, indicating the importance of voltage-operated Ca2+ channels and the Na(+)-K+ pump for this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between functional Na+ pumps and mitogenesis in cultured coronary artery smooth muscle cells.

An increase in functional sarcolemmal Na(+)-K(+)-ATPase (Na+ pump) precedes proliferation in vascular smooth muscle cells (VSMCs) seeded in 10% fetal bovine serum (FBS), but its role in mitogenesis is unresolved. Enzymatically dispersed canine coronary artery VSMCs were seeded in FBS and studied through confluence. Before a shift in cell cycle (G1-->S, G2 + M) and appearance of the nonmuscle isoform of myosin (MHCnm), intracellular Na+ content (Na+i) and cell volume (CV) increased (day 0 through day 3). Na+ pump number ([3H]-ouabain binding) increased at day 4 followed by a decrease in Na+i and CV. When Na+ pumps were inhibited by the addition of ouabain to FBS, VSMCs were arrested in G1, and MHCnm was not upregulated. Na+i increased similarly to that in FBS but failed to correct to day 0 levels. Withdrawal of ouabain at day 4 in culture led to an increase in Na+ pump number, a decrease in Na+i, entry of cells into S and G2 + M, and upregulation of MHCnm. These data suggest that Na+i, phenotypic modulation, and entry of cells into the cell cycle are temporally related, with Na+ pump-mediated correction of increased Na+i as a key event in the VSMC mitogenic process.

Effects of serotonin on intracellular pH and contraction in vascular smooth muscle.

Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.

Effects of pHi on Na(+)-H+, Na(+)-dependent, and Na(+)-independent C1(-)-HCO3-exchangers in vascular smooth muscle.

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.

Serotonin-induced Na+/K+ pump stimulation in vascular smooth muscle cells. Evidence for coupling to multiple receptor mechanisms.

Exposure of cultured canine femoral artery vascular smooth muscle cells to serotonin (5-HT) caused a 3.6-fold stimulation of ouabain-sensitive 86Rb uptake. The 5-HT2 receptor antagonist, ketanserin, partly blocked the 5-HT-mediated Na+/K+ pump stimulation and the 5-HT1/5-HT2 receptor antagonist, methiothepin, completely blocked the response, suggesting that both 5-HT1 and 5-HT2 receptors play a role in the 5-HT-mediated Na+/K+ pump activation. Second messengers generated by 5-HT2 receptor-mediated phosphoinositide hydrolysis, Ins(1,4,5)P3 and diacylglycerol were implicated in the stimulatory action of 5-HT on the vascular Na+/K+ pump. Like some other contractile agonists, 5-HT activated a Na+ influx pathway which caused Na+/K+ pump stimulation by increasing the rate-limiting substrate. The maximum stimulation of Na+ influx by 5-HT was 2.5-fold. The 5-HT-stimulated Na+ influx was totally blocked by methiothepin but only 29% inhibited by ketanserin, indicating that most of the Na+ influx was mediated by the 5-HT1 receptor. The 5-HT-stimulated Na+ influx was substantially inhibited by 50 microM dimethylamiloride, suggesting that the Na+ influx pathway stimulated by 5-HT was Na+/H+ exchange. BAPTA/AM 1,2-[bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester], an intracellular Ca++ chelator, partly blocked 5-HT-stimulated Na+ influx and ouabain-sensitive 86Rb uptake, suggesting that Ca++ is an important mediator of these responses. These data suggest that: 1) 5-HT, in addition to its well known activity as a contractile agonist, can stimulate the electrogenic Na+/K+ pump which, in theory, would tend to oppose contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Focal toxicity of oxysterols in vascular smooth muscle cell culture. A model of the atherosclerotic core region.

Cell necrosis and reactive cellular processes in and near the atherosclerotic core region might result from short-range interactions with toxic lipids. To model these interactions in cell culture, focal crystalline deposits of cholestane-3 beta,5 alpha,6 beta-triol, 25-OH cholesterol, and cholesterol were overlaid by a collagen gel, on which canine aortic smooth muscle cells were seeded. Oxysterols, but not cholesterol, caused focally decreased plating efficiency and cell death, leading to the formation of a persistent circular gap in the cell culture. Cholestanetriol was largely removed from the culture dishes over 3 to 4 weeks, whereas cholesterol and 25-OH cholesterol were largely retained. Smooth muscle cells were motile even in proximity to oxysterol crystals, with occasional suicidal migration toward the crystals. Chemoattraction, however, could not be demonstrated. Despite toxicity, cholestanetriol did not appear to alter the fraction of cells exhibiting 3H-thymidine uptake, even in areas close to the crystals. Thus, oxysterols may be toxic to some cells, without causing major impairment of the migration and proliferation of nearby cells. This would allow the simultaneous occurrence of cell death and proliferation evident in atherosclerosis.