Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.     

Differential development of carbachol-induced desensitization in receptor-mediated Ca2+ influx and Ca2+ release pathways in smooth muscle of guinea-pig taenia caeci

1. We have found that development of carbachol (CCh)-induced desensitization to receptor agonists, but not to receptor by-passed stimulation, is transiently interrupted by a Ca2+-dependent resensitization during the early stage in the smooth muscle of guinea-pig taenia caeci. To further characterize the receptor-mediated signal transduction pathways involved in this peculiar desensitization process, we examined the desensitization processes during Ca2+ influx- and Ca2+ release-mediated contractions in response to activation of muscarinic receptors or histamine H1 receptors. 2. Desensitization treatment with 10(-4) mol/L CCh for 30 min in the presence of extracellular Ca2+ resulted in desensitization to the muscarinic agonists McN-A-343 or AHR-602, which are known to induce contraction only in the presence of extracellular Ca2+ in taenia caeci. The development of desensitization to these agonists was interrupted by a transient resensitization at 1 min. In contrast, the transient resensitization phase was lost following removal of extracellular Ca2+ during the desensitization treatment with CCh; under these conditions, the desensitization developed gradually without an apparent resensitization phase. 3. Contractions to 10(-4) mol/L CCh and 10(-4) mol/L histamine in the absence of extracellular Ca2+ were gradually desensitized without a resensitization phase following the CCh desensitization treatment, irrespective of the presence or absence of extracellular Ca2+ during CCh treatment, although the onset of the desensitization was delayed under Ca2+-free conditions. 4. These results suggest that the receptor-mediated Ca2+ influx and Ca2+ release pathways are differentially desensitized to CCh and that the transient resensitization appears to regulate the desensitization process in response to Ca2+ influx-mediated contraction. Such differential processes of desensitization in receptor-mediated bifurcated signalling pathways may determine cellular responsiveness to certain types of stimuli, depending on the different Ca2+ sources required for contraction.     

Role of Ca2+ mobilization in muscarinic receptor-mediated membrane depolarization in guinea pig ileal smooth muscle cells

In single smooth muscle cells dispersed from guinea pig ileum, the muscarinic agonist carbachol (CCh) at 2 microM produced an oscillatory or sustained type of depolarization and at 100 microM, the latter type depolarization. Depletion of internal Ca2+ stores blocked the oscillatory response, but not the sustained responses to 2 microM and 100 microM CCh, although their decay after reaching the peak became faster. Blocking voltage-dependent Ca2+ channels (VDCCs) blocked both types of response to 2 microM CCh, but only slowed the initial rising phase of 100 microM CCh responses. Combination of Ca2+ store depletion and VDCC blockade abolished the responses to 2 microM CCh again and decreased those to 100 microM CCh in peak amplitude and persistency. Combination of Ca2+ store depletion with removal of extracellular Ca2+ markedly reduced or abolished the 100 microM CCh responses. The results suggest that muscarinic depolarization of the ileal cells requires Ca2+ mobilization for its generation and persistence; at weak muscarinic stimulation, both Ca2+ entry via VDCCs and Ca2+ release from internal stores may contribute to the Ca2+ mobilization; and under strong muscarinic stimulation, Ca2+ entry pathways resistant to VDCC blockers may also contribute to it.     

异紫堇啡碱对血管平滑肌钙内流和钙释放的影响

目的研究异紫堇啡碱（ＩＳＯＣ）对血管平滑肌钙内流和钙释放的影响，以初步阐明其作用方式。方法利用兔胸主动脉螺旋条标本观察ＩＳＯＣ对去甲肾上腺素（ＮＡ）及ＫＣｌ量效曲线的影响和对无钙液中ＮＡ、ＣａＣｌ２复钙、咖啡因缩血管效应的影响。结果ＩＳＯＣ１０μｍｏｌ·Ｌ－１对ＮＡ的量效曲线呈非竞争性拮抗作用，而对高钾的作用则不明显。在无钙液中，ＩＳＯＣ１００μｍｏｌ·Ｌ－１能明显抑制ＮＡ所致的收缩及复钙后外钙内流诱发的收缩，ＩＳＯＣ１０及３０μｍｏｌ·Ｌ－１则只作用于前者；各实验浓度的ＩＳＯＣ对咖啡因在无钙液中的缩血管作用均无影响。结论ＩＳＯＣ抑制受体中介的钙释放和钙内流，但不是典型的钙拮抗剂。     

BK channel activation by NS-1619 is partially mediated by intracellular Ca2+ release in smooth muscle cells of porcine coronary artery

1. Effects of NS-1619, an opener of large conductance Ca2+-activated K+ (BK) channel, on intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were examined in single myocytes freshly isolated from porcine coronary artery. 2. Under current clamp mode, the application of 1-30 microM NS-1619 hyperpolarized the membrane in concentration-dependent manner. The NS-1619-induced hyperpolarization was abolished by the presence of 100 nM iberiotoxin. 3. Application of 1-10 microM NS-1619 hyperpolarized the membrane by approximately 6 mV or less but did not change significantly the [Ca2+]i. When membrane hyperpolarization of 12 mV or so was caused by 30 microM NS-1619, [Ca2+]i was unexpectedly increased by approximately 200 nM. This increase in [Ca2+]i and the concomitant outward current activation were also observed under voltage-clamp at holding potential of -40 mV. 4. The increase in [Ca2+]i by 30 microM NS-1619 occurred mainly in peripheral regions than in the centre of the myocytes. The removal of extracellular Ca2+ affected neither the membrane hyperpolarization nor the increase in [Ca2+]i. 5. In the presence of 10 mM caffeine and 10 microM ryanodine, the increase in [Ca2+]i by 30 microM NS-1619 was not observed and the membrane hyperpolarization was reduced to approximately 67% of the control. 6. These results indicate that the opening of BK channels by NS-1619 at 30 microM, which is the most frequently used concentration of this agent, is partly due to Ca2+ release from caffeine/ryanodine-sensitive intracellular storage sites but is mainly due to the direct activation of the channels.     

Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release.

Neurotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig ileum, but was only 22% as effective as histamine. Neostigmine (0.1 micrometer) increased the effectiveness of neurotensin 4-fold. Atropine (0.1 micrometer) completely inhibited the contracting effect of neurotensin measured in the presence of neostigmine, whereas hexamethonium (0.1 micrometer) was without effect. It is concluded that in the longitudinal smooth muscle of the guinea-pig ileum, the contracting effect of neurotensin is mediated by the release of acetylcholine from nerve endings in response to a postganglionic stimulation of cholinergic nerve fibers.     

Hydroxylated xestospongins block inositol-1,4,5-trisphosphate-induced Ca2+ release and sensitize Ca2+-induced Ca2+ release mediated by ryanodine receptors

Inositol-1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) often coexist within the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane and coordinate precise spatial and temporal coding of Ca(2+) signals in most animal cells. Xestospongin C (XeC) was shown to selectively block IP(3)-induced Ca(2+) release and IP(3)R-mediated signaling (Gafni et al., 1997). We have further studied the specificity of xestospongin structures possessing ring hydroxyl (-OH) substituents toward IP(3)R, RyR, and ER/SR Ca(2+)-ATPase (SERCA) activities. XeC potently inhibits IP(3)R, weakly inhibits RyR1, and lacks activity toward SERCA1 and SERCA2. XeD (9-OH XeC), 7-OH-XeA, and araguspongin C isolated from the marine sponge Xestospongia species also inhibit IP(3)-mediated Ca(2+) release and lack activity toward SERCA. However, these hydroxylated derivatives possess a unique activity in that they enhance Ca(2+)-induced Ca(2+) release from SR vesicles by a mechanism involving the sensitization of RyR1 channels within the same concentration range needed to block IP(3)-induced Ca(2+) release. These results show that xestospongins and related structures lack direct SERCA inhibitory activity, as suggested by some previous studies. A new finding is that XeD and related structures possessing a hydroxylated oxaquinolizidine ring are IP(3)R blockers that also enhance Ca(2+)-induced Ca(2+) release mediated by RyRs. In intact cells, the actions of XeD are blocked by ryanodine pretreatment and do not interfere with thapsigargin-mediated Ca(2+) mobilization stemming from SERCA block. Hydroxylated bis-oxaquinolizadine derivatives isolated from Xestospongia species are novel bifunctional reagents that may be useful in ascertaining how IP(3)Rs and RyRs contribute to cell signaling.     

The binding of [3H]4-diphenylacetoxy-N-methylpiperidine methiodide to longitudinal ileal smooth muscle muscarinic receptors

Muscarinic receptors present in longitudinal ileum were characterized using the non-selective radioligand [3H]N-methylscopolamine [( 3H]NMS) and the M3 selective radioligand [3H]4-diphenylacetoxy-N-methylpiperidine methiodide [( 3H]4DAMP). In saturation studies, [3H]4DAMP, but not [3H]NMS, identified two populations of binding sites with 17% of the sites (155 fmol/mg protein) displaying high affinity (Kd = 0.39 nM) for [3H]4DAMP and the remaining sites displaying low affinity for the radioligand (Kd = 4.43 nM). In competition studies gallamine and methoctramine, but not AF-DX 116, identified two populations of [3H]NMS binding sites. Affinity estimates for gallamine and methoctramine indicated that 80% of the [3H]NMS binding sites were of the M2 subtype. The minor population of [3H]NMS binding sites could not be readily characterized, due partly to the low selectivity of the competing ligands and also to the relatively low density of the sites. In studies using the M3 muscarinic receptor selective radioligand [3H]4DAMP, the minor population of sites could be preferentially labeled by using a low concentration (0.4 nM) of [3H]4DAMP. Under these conditions, [3H]4DAMP labeled approximately equal levels of the two muscarinic receptor binding sites present in the ileum. Competition studies with AF-DX 116, gallamine and methoctramine indicated that the two [3H]4DAMP binding sites displayed the pharmacology expected of the M2 and M3 receptors, respectively. These results provide additional evidence that longitudinal ileal smooth muscle membranes contain both M2 and M3 muscarinic receptors and indicate that [3H]4DAMP is a useful ligand for identifying heterogeneity of muscarinic receptor subtypes.     

Acamprosate inhibits Ca2+ influx mediated by NMDA receptors and voltage-sensitive Ca2+ channels in cultured rat mesencephalic neurones

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-D-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to omega-conotoxin (omega-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 microM and 300 microM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of omega-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as omega-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.     

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.
2. α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED₅₀ values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.
3. Although the contraction caused by α,β-mATP was strongly diminished by Ca²⁺-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.
4. UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.
5. The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
6. In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.
7. These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.

     

Characterization of muscarinic receptors in guinea pig ileum longitudinal smooth muscle

Heterogeneity in the muscarinic receptor population of guinea pig ileum longitudinal smooth muscle was found in competition binding experiments against N-methyl[3H]scopolamine using either a cardioselective (AF-DX 116) or a smooth muscle-selective (hexahydrosiladifenidol) antimuscarinic compound. AF-DX 116 recognized 65% of the total receptors with high affinity and 35% with low affinity. Hexahydrosiladifenidol distinguished 24% of the total receptors with high affinity and 76% with low affinity. The two affinity binding constants displayed in smooth muscle by the compounds were similar to those of heart and glands, suggesting that the muscarinic receptor population in the smooth muscle is formed of about 30% glandular type and 70% cardiac type of the M2 receptors. In dissociation experiments, the rate of breakdown of the N-methyl[3H]scopolamine receptor complex in the smooth muscle was rapid and similar to the dissociation of N-methyl[3H]scopolamine from muscarinic receptors in cardiac membranes, supporting the evidence for the presence of a large fraction of the cardiac receptor type in smooth muscle. To further characterize the population of the smooth muscle receptors recognized as glandular type, we performed protection experiments with hexahydrosiladifenidol, which binds to glandular M3 receptors with high affinity. Smooth muscle membranes were initially incubated with this compound and then phenoxybenzamine was added to irreversibly alkylate the remaining unprotected receptors. Data from competition and dissociation binding experiments showed that, under these conditions, this protected fraction of the total receptor population in ileum smooth muscle had all the characteristics of the glandular type, i.e., slow N-methyl[3H]scopolamine dissociation and affinity constants for a series of selective and nonselective muscarinic antagonists in the same order of magnitude as those found in the glandular tissue. These findings, together with the known observation that hexahydrosiladifenidol is more potent in inhibiting the functional activation of muscarinic receptors in smooth muscle relative to heart, lead to the hypothesis that smooth muscle contractility is mediated by a muscarinic receptor subtype similar to that found in glandular tissue.     

Multiple muscarinic pathways mediate the suppression of voltage-gated Ca2+ channels in mouse intestinal smooth muscle cells

Background and purpose:

Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca²⁺ channel currents (I_Ca). However, little is known about which receptor subtype(s) mediate this effect.

Experimental approach:

The effect of carbachol on I_Ca was studied in single intestinal myocytes from M₂ or M₃ muscarinic receptor knockout (KO) and wild-type (WT) mice.

Key results:

In M₂KO cells, carbachol (100 µM) induced a sustained I_Ca suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I_Ca in M₃KO cells, but with a phasic time course. In M₂/M₃-double KO cells, carbachol had no effect on I_Ca. The extent of the suppression in WT cells was greater than the sum of the I_Ca suppressions in M₂KO and M₃KO cells, indicating that it is not a simple mixture of M₂ and M₃ receptor responses. The G_i/o inhibitor, Pertussis toxin, abolished the I_Ca suppression in M₃KO cells, but not in M₂KO cells. In contrast, the G_q/11 inhibitor YM-254890 strongly inhibited only the I_Ca suppression in M₂KO cells. Suppression of I_Ca in WT cells was markedly reduced by either Pertussis toxin or YM-254890.

Conclusion and implications:

In intestinal myocytes, M₂ receptors mediate a phasic I_Ca suppression via G_i/o proteins, while M₃ receptors mediate a sustained I_Ca suppression via G_q/11 proteins. In addition, another pathway that requires both M₂/G_i/o and M₃/G_q/11 systems may be operative in inducing a sustained I_Ca suppression.     

The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

Background and purpose:

The sarcoplasmic reticulum (SR) releases Ca²⁺ via inositol 1,4,5-trisphosphate receptors (IP₃R) in response to IP₃-generating agonists. Ca²⁺ release subsequently propagates as Ca²⁺ waves. To clarify the role of IP₃ production in wave generation, the contribution of a key enzyme in the production of IP₃ was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.

Experimental approach:

Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) measured using fluo-3. SR Ca²⁺ release was evoked either by activation of IP₃Rs (by carbachol or photolysis of caged IP₃) or ryanodine receptors (RyRs; by caffeine).

Key results:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto transients. The drug also inhibited [Ca²⁺]_cyto increases, evoked by direct IP₃R activation (by photolysis of caged IP₃) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca²⁺]_cyto and slowed the rate of Ca²⁺ removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP₃R- or RyR-mediated Ca²⁺ release.

Conclusions and implications:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto increases. However, the drug also reduced Ca²⁺ release when evoked by direct activation of IP₃R or RyR, slowed Ca²⁺ removal and increased steady-state [Ca²⁺]_cyto. These results suggest U-73122 reduces IP₃-evoked Ca²⁺ transients by inhibiting the SR Ca²⁺ pump to deplete the SR of Ca²⁺ rather than by inhibiting PI-PLC.     

Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle

1 In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N-[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca(2+) concentration ([Ca(2+)]c) with a higher EC(50) than that of the full agonist carbachol. The maximum response of [Ca(2+)]c or tension was not much different among the three agonists. The Ca(2+) channel blocker nicardipine markedly inhibited the effects of all three agonists 2 The contractile response to any agonist was antagonized in a competitive manner by M(2) receptor selective antagonists (N,N'-bis[6-[[(2-methoyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride and 11-[[2-[(diethlamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one), and the apparent order of M(2) antagonist sensitivity was McN-A343>pilocarpine>carbachol. M(3) receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M(3) antagonists behaved in a competitive manner in the case of the carbachol response. 3 McN-A343 failed to release Ca(2+) from the intracellular stores, and the Ca(2+)-releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca(2+) sensitivity of the contractile proteins. 4 McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. 5 The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca(2+) entry associated with the increased spike discharge and myofilaments Ca(2+) sensitization, and that Ca(2+) store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M(2) and M(3) receptors plays a crucial role in mediating the contraction response.     

Haloperidol differentiates smooth muscle contractions induced by release of intracellularly stored Ca and by influx of extracellular Ca.

1. The effects of haloperidol on smooth muscle contraction induced by carbachol, histamine, high K or caffeine in the presence or absence of extracellular Ca were investigated. 2. In the presence of extracellular Ca, the maximal contraction induced by carbachol was reduced by haloperidol, while that by histamine or high K was much less affected. 3. In Ca-free solution, contraction induced by histamine was extremely reduced by haloperidol, while that by carbachol was not affected. 4. These results suggest that haloperidol selectively inhibited signal transduction processes from activation of muscarinic acetylcholine receptors to influx of extracellular Ca and from activation of histamine H1-receptors to release of intracellularly stored Ca. 5. Caffeine-induced contraction in Ca-free solution was markedly potentiated by haloperidol, although haloperidol did not elicit contraction in Ca-free solution by itself. 6. These results suggest that haloperidol increased the sensitivity of Ca-induced Ca release channels to caffeine.     

Modulation of carbachol-induced [Ca2+]i oscillations by Ca2+ influx in single intestinal smooth muscle cells.

1. Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 microM), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor-coupled cationic current (Icat) in guinea-pig ileal smooth muscle cells by the whole cell patch-clamp technique. 2. Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh-induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations. 3. Application of nifedipine (1-3 microM) or D600 (2-5 microM) to block the voltage-gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at -20 mV, but it was without effect in cells held at -60 mV. 4. Displacement of the holding potential of -20 mV to -60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine-treated cells, the Icat oscillations varied in frequency voltage-dependently in a reverse and linear way within the range -80 to 40 mV. 5. Application of thapsigargin (1 or 2 microM), an inhibitor of Ca(2+)-ATPase in the membrane of internal Ca2+ stores, caused CCh-induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined. 6. The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh-induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.     

Elastase promotes aortic dilation by inhibiting Ca2+ influx into vascular smooth muscle

Abdominal aortic aneurysm (AAA) is a common vascular disease with, as of yet, unclear mechanism. Increased elastase activity and elastin degradation in the aorta are consistent findings in human AAA. Also, elastase perfusion of the aorta promotes aortic dilation in animal models of AAA. Although elastase-induced degradation of extracellular matrix proteins and the ensuing inflammation of the aortic wall have been implicated as possible causes of the aortic dilation in AAA, little is known regarding the effects of elastase on the mechanisms of aortic smooth muscle contraction. The purpose of this study was to test the hypothesis that elastase promotes aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats non-treated or treated with elastase. Initial experiments suggested that elastase alone caused matrix degradation. To avoid potential degradation of the extracellular matrix proteins by elastase, the same experiments were repeated in the presence of saturating concentrations of elastin (10 mg/ml). In normal Krebs (2.5 mM Ca2+), phenylephrine (Phe, 10(-5) M) caused contraction of the aortic strips that was significantly inhibited by elastase. The elastase-induced inhibition of Phe contraction was concentration- and time-dependent. At 5 U/ml elastase, the inhibition of Phe contraction was rapid in onset (2.4 +/- 0.3 minutes) and complete in 32 +/- 4 minutes. The inhibitory effects of elastase on Phe contraction were partially reversible. In Ca2+-free (2 mM EGTA) Krebs, Phe caused a small contraction that was not inhibited by elastase, suggesting that elastase does not inhibit Ca2+ release from the intracellular stores. Membrane depolarization by 96 mM KCl, which stimulates Ca2+ entry from the extracellular space, caused a contraction that was inhibited by elastase in a time-dependent and reversible fashion. The reversible inhibitory effects of elastase, particularly in the presence of saturating concentrations of elastin, suggest that they are not due to dissolution of the extracellular matrix or permanent damage to the smooth muscle contractile proteins. Elastase also caused significant inhibition of Phe- and KCl-induced 45Ca2+ influx. These data suggest that elastase promotes aortic relaxation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction, and thus further explain the role of increased elastase activity during the early development of AAA.     

蛋白激酶C与血管平滑肌α_1肾上腺素受体触发Ca~（2＋）内流的关系

在酶新鲜分离的犬肠系膜上动脉平滑肌细胞，蛋白激酶Ｃ（ＰＫＣ）的激活剂佛波二丁酯（ＰＤＢ）引起的胞内Ｃａ２＋增高作用可被ＰＫＣ抑制剂１－（５－异喹啉磺酰）－２－甲基哌嗪（Ｈ７）所阻断，而哌唑嗪和普萘洛尔则不能阻断ＰＤＢ的这一作用；１０μｍｏｌ·Ｌ－１苯福林引起的胞内Ｃａ２＋增高作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７部分阻断；在无Ｃａ２＋液，Ｈ７可部分抑制苯福林引起的内Ｃａ２＋释放和外Ｃａ２＋内流，两者分别被抑制了３３±３％和５８±６％；ＫＣｌ（２０－１００ｍｍｏｌ·Ｌ－１）可浓度依赖性地引起胞内钙升高，这一作用可被１０和２０μｍｏｌ·Ｌ－１Ｈ７不同程度地阻断；ＰＤＢ引起的胞内Ｃａ２＋增高也可分别被１．２５和２．５μｍｏｌ·Ｌ－１维拉帕米部分和全部阻断。上述结果提示ＰＫＣ参与苯福林引起的部分内Ｃａ２＋释放和外Ｃａ２＋内流，但以参与外Ｃａ２＋内流为主；这一作用可能与ＰＫＣ激活，引起电压依赖性Ｃａ２＋通道开放有关。