Selected contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca(2+) sensitivity.

To clarify the contribution of intracellular Ca(2+) concentration ([Ca(2+)](i))-dependent and -independent signaling mechanisms in arteriolar smooth muscle (aSM) to modulation of arteriolar myogenic tone by nitric oxide (NO), released in response to increases in intraluminal flow from the endothelium, changes in aSM [Ca(2+)](i) and diameter of isolated rat gracilis muscle arterioles (pretreated with indomethacin) were studied by fluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca(2+)](i) significantly increased and myogenic tone developed in response to elevations of extracellular Ca(2+) concentration. The Ca(2+) channel inhibitor nimodipine substantially decreased [Ca(2+)](i) and completely inhibited myogenic tone. Dilations to intraluminal flow (that were inhibited by N(omega)-nitro-L-arginine methyl ester) or dilations to the NO donor S-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were not accompanied by substantial decreases in aSM [Ca(2+)](i). 8-Bromoguanosine cGMP and the cGMP-specific phosphodiesterase inhibitor zaprinast significantly dilated arterioles yet elicited only minimal decreases in [Ca(2+)](i). Thus flow-induced endothelial release of NO elicits relaxation of arteriolar smooth muscle by a cGMP-dependent decrease of the Ca(2+) sensitivity of the contractile apparatus without substantial changes in the pressure-induced level of [Ca(2+)](i).     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Mechanisms of endothelial P2Y(1)- and P2Y(2)-mediated vasodilatation involve differential [Ca2+]i responses

The present study was designed to evaluate the role of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in the difference between P2Y(1)- and P2Y(2)-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca(2+) indicator, for simultaneous measurement of endothelial [Ca(2+)](i) and diameter. Luminal administration of 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), an endothelial P2Y(1) agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca(2+)](i) increases up to approximately 300 nM (resting [Ca(2+)](i) = 145 nM). UTP, an endothelial P2Y(2) agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca(2+)](i) increases to >400 nM. In the presence of N(G)-nitro-L-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca(2+)](i) threshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y(1) and P2Y(2) purinoceptors result in part from differential [Ca(2+)](i) responses. Consistent with this finding, these studies also demonstrate a higher [Ca(2+)](i) threshold for EDHF-dependent responses compared with NO.     

Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.     

EDHF,but not NO or prostaglandins,is critical to evoke a conducted dilation upon ACh in hamster arterioles

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or K(+) channels. Dilations upon ACh (71 +/- 3%) were conducted rapidly (<1 s) to upstream sites (at 1.4 mm: 37 +/- 5%). Although the NO donor SNP induced a similar local dilation (71 +/- 7%), this response was not conducted. Maximal amplitudes of ACh-induced dilations were not attenuated after inhibition of NO synthase and cyclooxygenase at the local and remote sites. However, additional treatment with a P-450 monooxygenase blocker (sulfaphenazole) strongly attenuated the local response (from 62 +/- 9 to 17 +/- 5%) and abrogated dilations at distant sites (at 0.67 mm: from 23 +/- 4% to 4 +/- 3%). Likewise, 17-octadecynoic acid strongly attenuated local and remote responses. Blockers of Ca(2+)-dependent K(+) channels (charybdotoxin or iberiotoxin) attenuated dilations at the local and remote sites after focal application at the ACh stimulation site. In marked contrast, treatment of the upstream site with these blockers was without any effect. We conclude that upon local stimulation with ACh, a cytochrome P-450 monooxygenase product is generated that induces local dilation via the activation of Ca(2+)-dependent K(+) channels and initiates conduction of the dilation. In contrast to the local site, neither activation of these K(+) channels nor the synthesis of NO or prostaglandins is necessary to dilate the arterioles at remote, distant sites. This suggests that endothelium-derived hyperpolarizing factor serves as an important mediator to initiate conducted dilations and, by doing so, may act as a key player in the coordination of arteriolar behavior in the microcirculatory network.     

Cerebral artery K(ATP)- and K(Ca)-channel activity and contractility: changes with development

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) channels (K(Ca)) play an important role in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca(2+)](i) in the absence and presence of selective K(+)-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the K(ATP)-channel opener pinacidil and the K(Ca)-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC(50)) = 5.0 +/- 0.1 and 8.2 +/- 0.1, respectively], with a modest decrease of [Ca(2+)](i). In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC(50) = 5.1 +/- 0.1 and 7.6 +/- 0.1, respectively) with no change in [Ca(2+)](i). In addition, the K(Ca)-channel blocker iberiotoxin (10(-7) to 10(-6) M) resulted in increased tension and [Ca(2+)](i) in both adult and fetal MCA, although the K(ATP)-channel blocker glibenclamide (10(-7) to 3 x 10(-5) M) failed to do so. Of interest, administration of 10(-7) M iberiotoxin totally eliminated vascular contraction and increase in [Ca(2+)](i) seen in response to 10(-5) M ryanodine. In precontracted fetal cerebral arteries, activation of the K(ATP) and K(Ca) channels significantly decreased both tension and [Ca(2+)](i), suggesting that both K(+) channels play an important role in regulating L-type channel Ca(2+) flux and therefore vascular tone in these vessels. In the adult, K(ATP) and the K(Ca) channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca(2+)](i). These channels show differing responses to inhibition, e.g., K(Ca)-channel inhibition, resulting in increased tension and [Ca(2+)](i), whereas K(ATP)-channel inhibition showed no such effect. In addition, the K(Ca) channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K(+)-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

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.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N(omega)-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195-H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is the initiating event and Ca(2+)-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca(2+)](i) and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca(2+)](i) increased from 69 +/- 8 nM to a peak value of 374 +/- 26 nM within 3 min and then declined to a sustained level at 190 +/- 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca(2+)](i). PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 +/- 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 +/- 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca(2+). These results provide direct evidence from intact microvessels that increased endothelial [Ca(2+)](i) is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.     

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.     

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.     

Ca(2+) signaling in porcine duodenal glands by muscarinic receptor activation

The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca(2+) concentration ([Ca(2+)](i)) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca(2+)](i), and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M(3) subtype far exceeded that for either M(1) or M(2) as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca(2+)](i) evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca(2+) deprivation. Caffeine had no effect on [Ca(2+)](i). ACh elicited Ca(2+)-dependent K(+) currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M(3) receptors linked to Ca(2+) release in porcine duodenal glands.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

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.     

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).