48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.     

Propagation of calcium waves along endothelium of hamster feed arteries

An increase in tissue blood flow requires relaxation of smooth muscle cells along entire branches of the resistance vasculature. Whereas the spread of hyperpolarization along the endothelium can coordinate smooth muscle cell relaxation, complementary signaling events have been implicated in the conduction of vasodilation. We tested the hypothesis that Ca(2+) waves propagate from cell to cell along the endothelium of feed arteries exhibiting spontaneous vasomotor tone. Feed arteries of the hamster retractor muscle were isolated, pressurized to 75 mmHg at 37 degrees C, and developed myogenic tone spontaneously. Smooth muscle cells and endothelial cells were loaded with the Ca(2+) indicator Fluo-4. An acetylcholine stimulus was delivered locally using microiontophoresis (1-microm pipette tip, 1 microA, 1 s), and Ca(2+) signaling within and along respective cell layers was determined using laser-scanning confocal microscopy. Acetylcholine triggered an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) of endothelial cells at the site of stimulation that preceded two distinct events: 1) a rapid synchronous decrease in smooth muscle [Ca(2+)](i) along the entire vessel and 2) an ensuing Ca(2+) wave that propagated bidirectionally along the endothelium at approximately 111 microm/s for distances exceeding 1 mm. Maximal dilation of vessels with either nifedipine (1 microM) or sodium nitroprusside (SNP, 100 microM) reduced the distance that Ca(2+) waves traveled to approximately 300 microm (P < 0.05). Thus Ca(2+) waves propagate along the endothelium of resistance vessels with vasomotor tone, and this signaling pathway is compromised during maximal dilation with nifedipine or SNP.     

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

Sympathetic nerve stimulation induces local endothelial Ca2+ signals to oppose vasoconstriction of mouse mesenteric arteries

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.     

A new technique for simultaneous and in situ measurements of Ca2+ signals in arteriolar smooth muscle and endothelial cells

We report here the first local and global Ca(2+) measurements made from in situ terminal arterioles. The advantages of the method are that there is minimal disturbance to the vessels, which retain their relationship to the tissue they are supplying (rat ureter) and the small size of vessel that can be studied. Good loading with the Ca(2+) indicator, Fluo-4 was obtained, and confocal sectioning through the tissue enabled vascular smooth muscle and endothelial cells to be clearly seen, along with red blood cells, nerve endings and the ureteric smooth muscle cells. We find the terminal arterioles to be extremely active, both spontaneously and in response to nor-adrenaline stimulation, with Ca(2+) sparks occurring in the vascular myocytes and Ca(2+) puffs in the endothelial cells. Even under resting conditions, endothelial cells produced oscillations and waves, which could pass from cell to cell, whereas the vascular myocytes only produced waves in response to agonist stimulation, and with no increase in the frequency of Ca(2+) sparks, and no spread from cell to cell. We compare our data to those obtained in dissected intact vessels and single cells. We conclude that this approach is a convenient and useful method for studying inter- and intracellular Ca(2+) signalling events and communication between cell types, particularly in very small vessels.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.     

Calcium signaling and oxidant stress in the vasculature

Recent evidence suggests that oxidant stress plays a major role in several aspects of vascular biology. Oxygen free radicals are implicated as important factors in signaling mechanisms leading to vascular pathologies such as postischemic reperfusion injury and atherosclerosis. The role of intracellular Ca(2+) in these signaling events is an emerging area of vascular research that is providing insights into the mechanisms mediating these complex physiological processes. This review explores sources of free radicals in the vasculature, as well as effects of free radicals on Ca(2+) signaling in vascular endothelial and smooth muscle cells. In the endothelium, superoxides enhance and peroxides attenuate agonist-stimulated Ca(2+) responses, suggesting differential signaling mechanisms depending on radical species. In smooth muscle cells, both superoxides and peroxides disrupt the sarcoplasmic reticulum Ca(2+)-ATPase, leading to both short- and long-term effects on smooth muscle Ca(2+) handling. Because vascular Ca(2+) signaling is altered by oxidant stress in ischemia-related disease states, understanding these pathways may lead to new strategies for preventing or treating arterial disease.     

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.     

Endothelium-dependent blunting of myogenic responsiveness after chronic hypoxia

Blunted agonist-induced vasoconstriction after chronic hypoxia is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and decreased vessel-wall Ca(2+) concentration ([Ca(2+)]). We hypothesized that myogenic vasoconstriction and pressure-induced Ca(2+) influx would also be attenuated in vessels from chronically hypoxic (CH) rats. Mesenteric resistance arteries isolated from CH [barometric pressure (BP), 380 Torr for 48 h] or normoxic control (BP, 630 Torr) rats were cannulated and pressurized. VSM cell resting membrane potential was recorded at intraluminal pressures of 40-120 Torr under normoxic conditions. VSM cells in vessels from CH rats were hyperpolarized compared with control rats at all pressures. Inner diameter was maintained for vessels from control rats, whereas vessels from CH rats developed less tone as pressure was increased. Pressure-induced increases in vessel-wall [Ca(2+)] were also attenuated for arteries from CH rats. Endothelium removal restored myogenic constriction to vessels from CH rats and normalized VSM cell resting membrane potential and pressure-induced Ca(2+) responses to control levels. Myogenic constriction and pressure-induced vessel-wall [Ca(2+)] increases remained blunted in the presence of nitric oxide (NO) synthase inhibition for arteries from CH rats. We conclude that blunted myogenic reactivity after chronic hypoxia results from a non-NO, endothelium-dependent VSM cell hyperpolarizing influence.     

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.     

Temperature effects on morphological integrity and Ca²⁺ signaling in freshly isolated murine feed artery endothelial cell tubes

To study Ca(2+) signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5-9 mo, 25-35 g). We tested the hypothesis that intracellular Ca(2+) concentration ([Ca(2+)](i)) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1-2 mm, width: 65-80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca(2+)](i) was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca(2+) K(d) values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca(2+)](i) remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ～220 nM at 24°C to ～500 nM at 32°C (P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca(2+)](i) increased by ～30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca(2+) signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca(2+) responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.     

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.     

Effects of chronic exercise on calcium signaling in rat vascular endothelium

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca(2+) signaling, we examined intracellular Ca(2+) concentration ([Ca(2+)](i)) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca(2+)](i) responses; 2) ACh induced heterogeneous [Ca(2+)](i) elevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca(2+)](i) elevation was inhibited by the Ca(2+) influx blocker SKF-96365, by a Ca(2+)-free buffer, or by high concentrations of extracellular K(+). We conclude that chronic exercise increases ACh-induced [Ca(2+)](i) elevation in rat aortic endothelium in situ, possibly by facilitating Ca(2+) influx.     

Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models

Normal pregnancy is associated with reductions in total vascular resistance and arterial pressure possibly due to enhanced endothelium-dependent vascular relaxation and decreased vascular reactivity to vasoconstrictor agonists. These beneficial hemodynamic and vascular changes do not occur in women who develop preeclampsia; instead, severe increases in vascular resistance and arterial pressure are observed. Although preeclampsia represents a major cause of maternal and fetal morbidity and mortality, the vascular and cellular mechanisms underlying this disorder have not been clearly identified. Studies in hypertensive pregnant women and experimental animal models suggested that reduction in uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia during late pregnancy may trigger the release of placental factors that initiate a cascade of cellular and molecular events leading to endothelial and vascular smooth muscle cell dysfunction and thereby increased vascular resistance and arterial pressure. The reduction in uterine perfusion pressure and the ensuing placental ischemia are possibly caused by inadequate cytotrophoblast invasion of the uterine spiral arteries. Placental ischemia may promote the release of a variety of biologically active factors, including cytokines such as tumor necrosis factor-alpha and reactive oxygen species. Threshold increases in the plasma levels of placental factors may lead to endothelial cell dysfunction, alterations in the release of vasodilator substances such as nitric oxide (NO), prostacyclin (PGI(2)), and endothelium-derived hyperpolarizing factor, and thereby reductions of the NO-cGMP, PGI(2)-cAMP, and hyperpolarizing factor vascular relaxation pathways. The placental factors may also increase the release of or the vascular reactivity to endothelium-derived contracting factors such as endothelin, thromboxane, and ANG II. These contracting factors could increase intracellular Ca(2+) concentrations ([Ca(2+)](i)) and stimulate Ca(2+)-dependent contraction pathways in vascular smooth muscle. The contracting factors could also increase the activity of vascular protein kinases such as protein kinase C, leading to increased myofilament force sensitivity to [Ca(2+)](i) and enhancement of smooth muscle contraction. The decreased endothelium-dependent mechanisms of vascular relaxation and the enhanced mechanisms of vascular smooth muscle contraction represent plausible causes of the increased vascular resistance and arterial pressure associated with preeclampsia.     

Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca(2+)]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100-200 microM ID) abluminally loaded with the Ca(2+) indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca(2+)] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca(2+)] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca(2+)] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca(2+)] contributes to attenuated reactivity following CH by decreasing VSM cell Ca(2+) sensitivity.