Ca2+ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction

Arteries that have developed myogenic tone (MT) are in a markedly different physiological state compared with those that have not, with higher cytosolic [Ca(2+)] and altered activity of several signal transduction pathways. In this study, we sought to determine whether alpha(1)-adrenoceptor-induced Ca(2+) signaling is different in pressurized arteries that have spontaneously developed MT (the presumptive physiological state) compared with those that have not (a common experimental state). At 32 degrees C and intraluminal pressure of 70 mmHg, cytoplasmic [Ca(2+)] was steady in most smooth muscle cells (SMCs). In a minority of cells (34%), however, at least one propagating Ca(2+) wave occurred. alpha(1)-Adrenoceptor activation (phenylephrine, PE; 0.1-10.0 microM) caused strong vasoconstriction and markedly increased the frequency of Ca(2+) waves (in virtually all cells). However, when cytosolic [Ca(2+)] was elevated experimentally in these arteries ([K(+)] 20 mM), PE failed to elicit Ca(2+) waves, although it did elevate [Ca(2+)] (F/F(0)) further and caused further vasoconstriction. During development of MT, the cytosolic [Ca(2+)] (F/F(0)) in individual SMCs increased, Ca(2+) waves disappeared (from SMCs that had them), and small Ca(2+) ripples (frequency approximately 0.05 Hz) appeared in approximately 13% of cells. PE elicited only spatially uniform increases in [Ca(2+)] and a smaller change in diameter (than in the absence of MT). Nevertheless, when cytosolic [Ca(2+)] and MT were decreased by nifedipine (1 microM), PE did elicit Ca(2+) waves. Thus alpha(1)-adrenoceptor-mediated Ca(2+) signaling is markedly different in arteries with and without MT, perhaps due to the elevated [Ca(2+)], and may have a different molecular basis. alpha(1)-Adrenoceptor-induced vasoconstriction may be supported either by Ca(2+) waves or by steady elevation of cytoplasmic [Ca(2+)], depending on the amount of MT.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Peroxynitrite diminishes myogenic tone in cerebral arteries: role of nitrotyrosine and F-actin

This study investigated the effect of peroxynitrite (OONO(-))-induced nitrosylation of filamentous (F)-actin on myogenic tone in isolated and pressurized posterior cerebral arteries (PCAs). Immunohistochemical staining was used to determine 3-nitrotyrosine (NT) and F-actin content in vascular smooth muscle after exposure to 10(-7) M or 10(-4) M OONO(-) for 5 or 60 min in isolated third-order PCAs (n = 37) from male Wistar rats pressurized to 75 mmHg in an arteriograph chamber, quantified with confocal microscopy. Additionally, the role of K(+) channels in OONO(-)-induced dilation was investigated with 3 microM glibenclamide or 10 mM tetraethylammonium chloride before OONO(-) exposure. OONO(-) (10(-4) M) induced a 40% dilation of tone (P < 0.05) while diminishing F-actin content by half (P < 0.05) and causing a 60-fold increase in NT (P < 0.05) in the vascular smooth muscle of PCAs. Additionally, F-actin was inversely correlated with both diameter and NT content (P < 0.05) and was significantly colocalized in the vascular smooth muscle with NT (overlap coefficient = 0.8). The dilation to ONOO(-) was independent of K(+) channel activity and thiol oxidation as glibenclamide, tetraethylammonium chloride, and dithiothreitol had no effect on OONO(-)-induced dilation or F-actin or NT content in PCAs. Because NT was colocalized with F-actin, we hypothesize that OONO(-) induces nitrosylation of F-actin in vascular smooth muscle leading to depolymerization and the subsequent loss of myogenic tone, which may promote vascular damage during oxidative stress such as in ischemia and reperfusion injury.     

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.     

Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Mg2+-Ca2+ interaction in contractility of vascular smooth muscle: Mg2+ versus organic calcium channel blockers on myogenic tone and agonist-induced responsiveness of blood vessels

Contractility of all types of invertebrate and vertebrate muscle is dependent upon the actions and interactions of two divalent cations, viz, calcium (Ca2+) and magnesium (Mg2+) ions. The data presented and reviewed herein contrast the actions of several organic Ca2+ channel blockers with the natural, physiologic (inorganic) Ca2+ antagonist, Mg2+, on microvascular and macrovascular smooth muscles. Both direct in vivo studies on microscopic arteriolar and venular smooth muscles and in vitro studies on different types of blood vessels are presented. It is clear from the studies done so far that of all Ca2+ antagonists examined, only Mg2+ has the capability to inhibit myogenic, basal, and hormonal-induced vascular tone in all types of vascular smooth muscle. Data obtained with verapamil, nimopidine, nitrendipine, and nisoldipine on the microvasculature are suggestive of the probability that a heterogeneity of Ca2+ channels, and of Ca2+ binding sites, exists in different microvascular smooth muscles; although some appear to be voltage operated and others, receptor operated, they are probably heterogeneous in composition from one vascular region to another. Mg2+ appears to act on voltage-, receptor-, and leak-operated membrane channels in vascular smooth muscle. The organic Ca2+ channel blockers do not have this uniform capability; they demonstrate a selectivity when compared with Mg2+. Mg2+ appears to be a special kind of Ca2+ channel antagonist in vascular smooth muscle. At vascular membranes it can (i) block Ca2+ entry and exit, (ii) lower peripheral and cerebral vascular resistance, (iii) relieve cerebral, coronary, and peripheral vasospasm, and (iv) lower arterial blood pressure. At micromolar concentrations (i.e., 10-100 microM). Mg2+ can cause significant vasodilatation of intact arterioles and venules in all regional vasculatures so far examined. Although Mg2+ is three to five orders of magnitude less potent than the organic Ca2+ channel blockers, it possesses unique and potentially useful Ca2+ antagonistic properties.     

Mg2+-induced changes of lipid order and conformation of (Na+ + K+)-ATPase

The effect of magnesium on the phospholipid order parameter and not the conformation of purified pig kidney outer medulla (Na+ + K+)-ATPase was investigated by fluorescence techniques. Measurements with a fluorescent probe TMA-DPH and its sensitized fluorescence with tryptophan residues as donors revealed that magnesium increased the order of the membrane phospholipids both in the lipid annulus and in the bulk phase. Changes in the lipid order induced by Mg2+ can be closely referred to the protein arrangement followed by the steady-state anisotropy of FITC-labeled (Na+ + K+)-ATPase.     

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase

The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Role of Ba2+-resistant K+ channels in endothelium-dependent hyperpolarization of rat small mesenteric arteries

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.     

Increased myogenic responses in uterine but not mesenteric arteries from pregnant offspring of diet-restricted rat dams

Results of epidemiological and animal studies suggest a link between poor in utero growth and cardiovascular disease in adult offspring. Few studies, however, have examined the effects of maternal undernutrition on the vasculature of pregnant female offspring, and to our knowledge, no studies have examined myogenic responses, which are essential to vascular tone development, in these animal models. Thus, myogenic responses were assessed in radial uterine arteries of pregnant female offspring to determine if diet restriction during pregnancy could contribute to transgenerational effects. These results were compared to those in mesenteric arteries, which greatly contribute to peripheral vascular resistance. Myogenic responses in the presence and absence of inhibitors for nitric oxide synthase (NOS) and prostaglandin H synthase (PGHS) were measured in arteries isolated from pregnant, 3-mo-old female offspring of control-fed (C(off)) and globally diet-restricted (DR(off)) rat dams. Although no differences were found in pregnancy weight gain, litter size, or fetal weights, placental size was significantly reduced in DR(off) compared to C(off). Enhanced myogenic reactivity was observed at the highest pressure tested (110 mm Hg) in uterine, but not in mesenteric, arteries from DR(off) compared to C(off). Inhibition of NOS, but not of PGHS, significantly increased myogenic responses in uterine arteries at pressures greater than 80 mm Hg in C(off) but, interestingly, not in DR(off) compared to untreated uterine arteries. Thus, impaired uterine vascular function in diet-restricted pregnant rat dams, which leads to similar impairment in their pregnant offspring, may be a mechanism through which transgenerational effects of unhealthy pregnancies occur.     

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.     

Blockade of Ca2+ channels inhibits K+ contractures but not twitches in skeletal muscle

The effects of the voltage-sensitive, calcium channel blocking agents, D-600 and verapamil, on twitches and K+-induced contractures were studied using frog's toe muscles. K+-contracture tension was reduced by concentrations as low as 10(-8) M and the contractures were blocked by 10(-6) M. There was no significant difference in the effects of the two drugs. Twitches were potentiated by 5 X 10(-5) M D-600 and blocked only at 3 X 10(-4) M. The latter concentration also produced contractures in the toe muscles. As shown by other workers, the higher concentration also blocks action potential production and this is probably the way in which it blocks the twitch. Raising the bathing solution Ca2+ concentration from 1.08 to 10 or 20 mM, produced only a small, inconsistent, noncompetitive antagonism of the D-600 block of K+ contractures.     

The role of sphingosine kinase 1/sphingosine-1-phosphate pathway in the myogenic tone of posterior cerebral arteries

Aims

The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.

Methods and Results

We measured pressure-induced myogenic response, Ca²⁺ concentration, and 20 kDa myosin light chain phosphorylation (MLC₂₀) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca²⁺ and in the presence of mechanosensitive ion channel and voltage-sensitive Ca²⁺ channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca²⁺ concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca²⁺ concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC₂₀ phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC₂₀ phosphorylation. The pressure-induced increase of MLC₂₀ phosphorylation was inhibited by pre-treatment of arteries with SKI.

Conclusions

Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.     

Transient increases in diameter and [Ca(2+)](i) are not obligatory for myogenic constriction

Studies were performed to determine the significance of temporal variation in vascular smooth muscle Ca(2+) signaling during acute arteriolar myogenic constriction and, in particular, the importance of the stretch-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) transient in attaining a steady-state mechanical response. Rat cremaster arterioles (diameter approximately 100 microm) were dissected from surrounding tissues, and vessel segments were pressurized in the absence of intraluminal flow. For [Ca(2+)](i) measurements, vessels were loaded with fura 2 and fluorescence emitted by excitation at 340 and 380 nm was measured using video-based image analysis. Ca(2+) and diameter responses were examined after increases in intravascular pressure were applied as an acute step increase or a ramp function. Additional studies examined the effect of longitudinal vessel stretch on [Ca(2+)](i) and arteriolar diameter. Step increase in intraluminal pressure (from 50 to 120 mmHg) caused biphasic change in [Ca(2+)](i) and diameter. [Ca(2+)](i) transiently increased to 114.0 +/- 2.0% of basal levels and subsequently declined to 106.7 +/- 4.4% at steady state. Diameter initially distended to 125.4 +/- 2.1% of basal levels before constricting to 71.1 +/- 1.2%. In contrast, when the same pressure increase was applied as a ramp function (over 5 min) transient vessel distension and transient increase in [Ca(2+)](i) were prevented, yet at steady state vessels constricted to 71.3 +/- 2.5%. Longitudinal stretch resulted in a large [Ca(2+)](i) transient (158 +/- 19% of basal) that returned to baseline despite maintenance of the stretch stimulus. The data demonstrate that the initial vessel distension (reflecting myocyte stretch) and associated global [Ca(2+)](i) transient are not obligatory for myogenic contraction. Thus, although arteriolar smooth muscle cells are responsive to acute stretch, the resulting changes in myogenic tone may be more closely related to other mechanical variables such as wall tension.     

Macrophage arachidonate-mobilizing phospholipase A2: role of Ca2+ for membrane binding but not for catalytic activity.

A recently purified Ca(2+)-dependent intracellular phospholipase A2 from spleen, kidney and macrophage cell lines is activated by Ca2+ at concentrations achieved intracellularly. Using enzyme from the murine cell line J774 we here demonstrate the formation of a ternary complex of phospholipase, 45Ca2+ and phospholipid vesicle, and provide evidence for a single Ca(2+)-binding site on the enzyme involved in its vesicle binding. Although Ca2+ binds to and functions as an activator of the enzyme, this ion does not appear to be involved in its catalytic mechanism, since enzyme brought to the phospholipid vesicle by molar concentrations of NaCl or NH4+ salts exhibited Ca(2+)-independent catalytic activity.     

Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.     

The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells

We have studied the role of Mg2+ in the inactivation of inwardly rectifying K+ channels in vascular endothelial cells. Inactivation was largely eliminated in Mg(2+)-free external solutions and the extent of inactivation was increased by raising Mg2+o. The dose-response relation for the reduction of channel open probability showed that Mg2+o binds to a site (KD = approximately 25 microM at -160 mV) that senses approximately 38% of the potential drop from the external membrane surface. Analysis of the single-channel kinetics showed that Mg2+ produced a class of long-lived closures that separated bursts of openings. Raising Mg2+o reduced the burst duration, but less than expected for an open-channel blocking mechanism. The effects of Mg2+o are antagonized by K+o in manner which suggests that K+ competes with Mg2+ for the inactivation site. Mg2+o also reduced the amplitude of the single-channel current at millimolar concentrations by a rapid block of the open channel. A mechanism is proposed in which Mg2+ binds to the closed channel during hyperpolarization and prevents it from opening until it is occupied by K+.     

Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force

The nephrotoxic metal Cd²⁺ causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K⁺ cycle involving a K⁺ uniporter and a K⁺/H⁺ exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd²⁺ on K⁺ cycling in rat kidney cortex mitochondria. Cd²⁺ (EC₅₀ 19 µM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd²⁺-induced swelling of energized mitochondria had a similar EC₅₀ value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca²⁺ uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd²⁺ influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (_m) induced by K⁺ influx did not prevent contraction, but addition of the K⁺/H⁺ exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 µM), abolished contraction, suggesting the mitochondrial pH gradient (pH_m) driving contraction. Accordingly, a quinine-sensitive partial dissipation of pH_m was coincident with the swelling-contraction phase. The data indicate that Cd²⁺ enters the matrix through the MCU to activate a K⁺ cycle. Initial K⁺ load via a Cd²⁺-activated K⁺ uniporter in the IMM causes osmotic swelling and breakdown of _m and triggers quinine-sensitive K⁺/H⁺ exchange and contraction. Thus Cd²⁺-induced activation of a K⁺ cycle contributes to the dissipation of the mitochondrial protonmotive force. bongkrekic acid; cyclosporin A; lanthanum; Ru360; ruthenium red     

Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.