C. van Breemen

Ion channels and regulation of intracellular calcium in vascular endothelial cells.

Endothelial cells in vivo form an interface between flowing blood and vascular tissue, responding to humoral and physical stimuli to secrete relaxing and contracting factors that contribute to vascular homeostasis and tone. The activation of endothelial cell‐surface receptors by vasoactive agents is coupled to an elevation in cytosolic Ca2+, which is caused by Ca2+ entry via ion channels in the plasma membrane and by Ca2+ release from intracellular stores. Ca2+ entry may occur via four different mechanisms: 1) a receptor‐mediated channel coupled to second messengers; 2) a Ca2+ leak channel dependent on the electrochemical gradient for Ca2+; 3) a stretch‐activated nonselective cation channel; and 4) internal Na+‐dependent Ca2+ entry (Na+‐Ca2+ exchange). The rate of Ca2+ entry through these ion pathways can be modulated by the resting membrane potential. Membrane potential may be regulated by at least two types of K channels: inwardly rectifying K channels activated upon hyperpolarization or shear stress; and a Ca2+‐activated K channel activated upon depolarization, which may function to repolarize the agonist‐stimulated endothelial cell. After agonist stimulation, cytosolic Ca2+ increases in a biphasic manner, with an initial peak due to inositol 1,4,5‐trisphosphate‐mediated Ca2+ release from intracellular stores, followed by a sustained plateau that is dependent on the presence of [Ca2+]o and on membrane potential. The delay in agonist‐activated Ca2+ influx is consistent with the coupling of receptor activation to Ca2+ entry via a second messenger. Oscillations in [Ca2+]i, which may involve both Ca2+ entry and release, have been observed in isolated and confluent endothelial cell monolayers stimulated by histamine and bradykinin. Receptor‐mediated Ca2+ entry, release, and refilling of intracellular stores follows a cycle that involves the plasma membrane.—Adams, D. J.; Barakeh, J.; Laskey, R.; Van Breemen, C. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J. 3: 2389‐2400; 1989.

Calcium requirement for activation of intact aortic smooth muscle

1. Elevation of [K+]e induced a contraction of rabbit aorta. If 10 m M‐La3+ was applied to rabbit aortae prior to [K+]e elevation no contraction occurred. When 10 m M‐La3+ was applied simultaneously with, or at short time periods after, elevation of [K+]e graded contractions were obtained whose magnitudes were higher if La3+ was added at a later stage.

Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle.

The purpose of this study was to investigate the effects of the intracellular messenger cyclic GMP (cGMP) on sequestration of cytosolic calcium (Ca2+) into the intracellular Ca2+ store (the sarcoplasmic reticulum) of vascular smooth muscle. Using saponin-skinned primary cultures of rat aortic smooth muscle, we investigated the effect of cGMP on 45Ca uptake in monolayers of cells. The intracellular store was loaded with Ca2+ by exposing the skinned cells to a 45Ca-labeled 1-p.M free Ca2+-containing solution for varying durations (0-20 minutes). Addition of 10 μM cGMP to six monolayers increased both the initial Ca2+ uptake at 2 minutes (control, 240 ±8 pmol Ca2+/104 cells; + cGMP 295 ±7; mean±SEM; n = 6, p<0.01) and the final steady-state uptake reached at 20 minutes (control, 0.96±0.03 nmol Ca2+104 cells; + cGMP 1.12±0.03, p<0.02). This stimulation of uptake was quantitatively similar to that caused by 10 μM cyclic AMP. It occurred at varying ambient cytosolic Ca2+ concentrations (0.1−1.0 μM Ca2+) and was not further enhanced by addition of 10 μM cGMP-dependent protein kinase. The dose-response of stimulation of Ca2+ uptake with cGMP indicated an EDn of 5 nM cGMP. The release of Ca2+ from the sarcoplasmic reticulum in response to inositol 1, 4, 5-trisphosphate or caffeine was unaffected by cGMP. We conclude that the relaxation of vascular smooth muscle with cGMP-producing vasodilators is mediated in part by sequestration of cytosolic Ca2+ by the sarcoplasmic reticulum.

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells.

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.

The mechanism of alpha-adrenergic activation of the dog coronary artery

Norepinephrine (NE) activates isolated coronary conduit arteries by stimulating Ca2+ uptake into the smooth muscle cells. Blockade of Ca2+ influx by removal of Ca2+ from the bathing medium or addition of 10 mM LaCl3 prevents the NE-induced contraction in the dog coronary artery but still allows NE to induce a rapid transient contraction in the rabbit aorta. Under these conditions, NE stimulates 45Ca2+ efflux from rabbit aorta but fails to do so in the coronary artery. The difference in behaviour between the two arteries is attributed by the presence of an intracellular NE-sensitive Ca2+ fraction in the rabbit aorta and its absence from the dog coronary artery. This difference also explains the much greater sensitivity of the NE-induced contractions in the dog coronary to the relaxant effects of the Ca2+ antagonists, D600 and SKF525A, than that seen in the rabbit aorta. High K+-induced contractions of both the coronary artery and the aorta are equally sensitive to the Ca2+ antagonists.

Mechanism of Ca++ antagonist-induced vasodilation. Intracellular actions.

We studied the effects of Ca++ antagonists on intact and skinned muscles of rabbit mesenteric artery. Intact muscle contractions were inhibited by 10−6M diltiazem, whereas greater levels were required to abolish contractions in skinned muscle fibers. In contrast, nisoldipine had no effect on skinned muscle contractions, although it inhibited, almost completely, the contraction of intact muscle at concentrations below 10−6M. In the presence of EGTA, norepinephrine-induced contractions result from a release of Ca++ from an intracellular store. Diltiazem inhibited these contractions at concentrations between 10−6 and 10−4M. Higher doses were required in studies with skinned muscle preparations. Unlike diltiazem, nisoldipine only partially inhibited the Ca++-free norepinephrine-induced contractions in the range of 10−7 to 10−5 M. From these results, we assumed that at low concentrations (below 10−6 M), diltiazem induced relaxation by blocking Ca++ influx, whereas at relatively high concentrations (above 10−6 M), an inhibition of Ca++ release from an intracellular store also occurred. A similar conclusion was reached regarding the mechanism whereby nisoldipine inhibits force developments.

Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery.

This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions in Staphylococcus alpha-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with alpha-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 microM GTP significantly enhanced myofilament Ca2+ sensitivity as compared with the addition of Ca2+ alone (EC50, 0.47 microM Ca2+ for Ca2+ alone and 0.13 microM Ca2+ for ET-1 plus (GTP). This enhanced sensitivity was reversed by GDP beta S. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 microM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 microM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 microM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+ dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+ sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells.

The relative importance of intracellular and extracellular Ca2+ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca2+ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC50 of 4×10−10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca2+. However, in the absence of external Ca2+, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with45Ca, bradykinin increased the45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in45Ca efflux induced by bradykinin of 1.3×10−9 M. The involvement of an intracellular Ca2+ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca2+,Mg2+-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca2+ concentrations showed at least two compartments involved in the uptake of Ca2+. The45Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the45Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP3). The IP3-induced release was not affected by apyrase or exogenous GTP. The EC50 for the release of Ca2+ by IP3 was 1.0 μM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca2+ influx and the release of intracellular Ca2+. The release of calcium from one of the high affinity intracellular Ca2+ stores is mediated by the intracellular second messenger, IP3.

ATP stimulates calcium influx in primary astrocyte cultures

The effect of ATP and other purines on 45Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced 45Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

Differences in norepinephrine activation and diltiazem inhibition of calcium channels in isolated rabbit aorta and mesenteric resistance vessels.

The mechanisms of norepinephrine stimulation of calcium ion entry in isolated rabbit aorta and mesenteric resistance vessels were studied through measurements of effects on calcium-45 influx, tension, and membrane potential. The resistance vessels were considerably less sensitive to norepinephrine than the aorta. The aorta exhibited complex dose-response curves for norepinephrine-stimulated calcium influx and contraction, whereas these were simple in the arterioles. Both vessels were depolarized with increasing concentrations of potassium. Norepinephrine did not depolarize the aorta, whereas it did depolarize the mesenteric resistance vessels. This result supports the contention that norepinephrine opens receptor-operated channels to induce calcium entry in the aorta, while it may activate potential sensitive calcium channels in the mesenteric resistance vessels. However, the maximum depolarization with norepinephrine (10−4 m) in the arterioles was completely blocked by 10−5 m diltiazem, whereas that induced by 80 Mm potassium was unaltered by the diltiazem. Furthermore, 10−4 m norepinephrine was able to stimulate virtually the same contraction and calcium influx in 80 mM potassium-depolarized arterioles as in normal polarized tissues. These results are consistent with norepinephrine opening of receptor-operated channels to allow calcium entry in the rabbit mesenteric resistance vessels. That the behavior of norepinephrine-activated channels in the aorta is more complex than in the arterioles is further illustrated by a dramatically decreasing sensitivity of norepinephrine-stimulated calcium influx to diltiazem with increasing norepinephrine in the aorta but not in the arterioles. We have hypothesized that the complexity of the norepinephrine-stimulated calcium influx in the aorta compared to the resistance vessels may be related to the substantial release of intracellular calcium in the former, such that, as release of intracellular calcium is increased, sensitivity of norepinephrine-activated channels to diltiazem decreases.