Dynamics of Ca2 handling in vascular smooth muscle

Intracellular Ca2+ is a key regulator of vascular contractility, and thereby of blood perfusion and pressure. Ca2+ signals are essential also for cell migration, proliferation and regulation of numerous enzymes. Besides producing vascular contraction smooth muscle cells may also modulate to a synthetic phenotype and proliferate as an initial stage in the atherosclerotic process. In this work we show that altered Ca2+ handling is an early step in phenotypic modulation, which might be important for promoting or regulating the process. Using tissue culture of rat tail and basilar arteries, large up-regulation of intracellular Ca2+ stores and of 'store operated' Ca2+ influx was evident, before changes in differentiation markers for the contractile state occurred. Further increase in intracellular Ca2+ storage could be induced by increasing the external load of Ca2+ during the culture period. The diverse roles of Ca2+ in signalling require mechanisms for selectivity, which appear to involve spatial and temporal coding. Vascular smooth muscle exhibits intracellular Ca2+ 'waves' and 'sparks'. Using culture in the presence of ryanodine, we achieved preparations lacking Ca2+ release from intracellular stores through the ryanodine receptor, but with intact inositol 1,4,5-trisphosphate sensitive release. These vessels lacked Ca2+ sparks but showed normal wave activity, indicating selective roles of the two modes of Ca2+ release for these different kinds of signals. Inhibition of metabolism caused an increase in wave frequency and a reduction in amplitude, with no effect on averaged tissue [Ca2+]i, or myosin phosphorylation. This suggests that alteration of the wave pattern might regulate contractile force produced by summation of asynchronous phasic contractions of individual cells. Many receptors, enzymes and signalling molecules are concentrated in cholesterol-rich membrane regions (caveolae), which thus may be important for spatial coding. Extraction of cholesterol from caveolae had little effect on wave generation, but disrupted the coupling from many cell membrane receptors to contraction

Contractile effects of polycations in permeabilized smooth muscle

The polycations spermine, neomycin and polylysine potentiated Ca(2+)-activated force in beta-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in chi-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by beta-escin, which eliminated the contractile effect of GTPgammaS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca(2+)-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca(2+)-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca(2+)-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40 micron. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR in inhibit phosphatase activity was increased by the polycations, but only at [Ca2+] < 0.3 micron. The results suggest that polycations increase Ca(2+)-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) or D-myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 microM), 5-HT (10 microM), or AVP (0.1 microM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 microM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]i and involving ryanodine- but not IP3 receptor-mediated Ca2+ release

Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca(2+) handling

1. The roles of intracellular Ca(2+) stores and ryanodine (Ry) receptors for vascular Ca(2+) homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 – 100 μM) for up to 4 days. 2. Acute exposure to Ry or the non-deactivating ryanodine analogue C(10)-O(eq) glycyl ryanodine (10 μM) eliminated Ca(2+) release responses to caffeine (20 mM) and noradrenaline (NA, 10 μM), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to Ry. 3. Ry receptor protein was detected on Western blots in arteries cultured either with or without Ry. 4. Brief Ca(2+) release events (sparks) were absent after culture with Ry, whereas Ca(2+) waves still occurred. The propagation velocity of waves was equal (∼19 μm s(−1)) in tissue cultured either with or without Ry. 5. Inhibition of Ca(2+) accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 μM) decreased contractility due to Ca(2+)-induced cell damage. In contrast, culture with Ry did not affect contractility. 6. Removal of Ca(2+) from the cytosol following a Ca(2+) load was retarded after Ry culture. Thapsigargin reduced the rate of Ca(2+) removal in control cultured rings, but had no effect after Ry culture. 7. It is concluded that intracellular Ca(2+) stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca(2+) sparks and for SR-dependent recovery from a Ca(2+) load, but not for Ca(2+) waves or basal Ca(2+) homeostasis

Effects of oxygen tension on energetics of cultured vascular smooth muscle.

Chronic hypoxia is a clinically important condition known to cause vascular abnormalities. To investigate the cellular mechanisms involved, we kept rings of a rat tail artery for 4 days in hypoxic culture (HC) or normoxic culture (NC) (PO(2) = 14 vs. 110 mmHg) and then measured contractility, oxygen consumption (JO(2)), and lactate production (J(lac)) in oxygenated medium. Compared with fresh rings, basal ATP turnover (J(ATP)) was decreased in HC, but not in NC, with a shift from oxidative toward glycolytic metabolism. JO(2) during mitochondrial uncoupling was reduced by HC but not by NC. Glycogen stores were increased 40-fold by HC and fourfold by NC. Maximum tension in response to norepinephrine and the JO(2) versus tension relationship (JO(2) vs. high K(+) elicited force) were unaffected by either HC or NC. Force transients in response to caffeine were increased in HC, whereas intracellular Ca(2+) wave activity during adrenergic stimulation was decreased. Protein synthesis rate was reduced by HC. The results show that long-term hypoxia depresses basal energy turnover, impairs mitochondrial capacity, and alters Ca(2+) homeostasis, but does not affect contractile energetics. These alterations may form a basis for vascular damage by chronic hypoxia

Increased store-operated Ca2+ entry into contractile vascular smooth muscle following organ culture

Ca2+ inflow via store-operated Ca2+ channels was investigated in rings of rat tail and basilar arteries kept in serum-free organ culture, which is known to preserve the contractility of the vascular smooth muscle. After culture for 3-4 days, Ca2+ release from intracellular stores in response to caffeine (20 mM) was augmented 2- to 4-fold. Following depletion of intracellular Ca2+ stores by caffeine and thapsigargin (10 microM), addition of Ca2+ (2.5 mM) caused an increase in the intracellular Ca2+ concentration which was 2-3 times greater in cultured than in freshly dissected rings, and was not affected by verapamil (10 microM). In contrast, L-type Ca2+ channel currents were decreased by 20% after culture. While freshly dissected rings developed no or very little force in response to the addition of Ca2+ after store depletion, cultured rings developed 42% (tail artery) and 60% (basilar artery) of the force of high-K+-induced contractions. These contractions in cultured vessels were insensitive to verapamil but could be completely relaxed by SKF-96365 (30 microM). Store depletion by caffeine increased the Mn2+ quench rate 3- to 4-fold in freshly dissected as well as cultured tail artery, while there was no increase in freshly dissected basilar artery, but a 3-fold increase in cultured basilar artery. Uptake of Ca2+ into intracellular stores was twice as rapid in cultured as in freshly dissected tail artery. This study shows that organ culture of vascular smooth muscle tissue causes changes in Ca2+ handling, resembling the pattern seen in dedifferentiating smooth muscle cells in culture, although contractile properties are maintained