Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes

In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP₂) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP₂, a water soluble form of PIP₂, to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP₂-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP₂ antibodies and by depletion of tissue PIP₂ levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP₂ to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP₂ at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP₂ whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP₂. Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP₂ can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP₂ in SOC activation requires PKC-dependent phosphorylation of TRPC1.     

Activation of store-operated channels by noradrenaline via protein kinase C in rabbit portal vein myocytes

In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca²⁺-permeable store-operated channels (SOCs) by noradrenaline in rabbit portal vein smooth muscle cells. With cell-attached recording, bath application of noradrenaline, 1-oleoyl-acetyl- sn -glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu) evoked single channel currents. The biophysical properties of these channel currents were similar to those of the channel currents activated by depletion of internal Ca²⁺ stores with cyclopiazonic acid (CPA). The activation of SOCs in cell-attached recording by noradrenaline, OAG, PDBu, CPA and the acetoxymethyl ester form of BAPTA (BAPTA-AM) was markedly inhibited by the PKC inhibitors chelerythrine and RÖ-31-8220. In isolated outside-out patches CPA did not evoke SOCs but noradrenaline stimulated SOC activity, which was reduced by about 90 % by PKC inhibitors. The addition of the serine/threonine phosphatase inhibitors calyculin A and microcystin also stimulated SOCs in isolated outside-out patches. It is concluded that in rabbit portal vein myocytes, noradrenaline activates SOCs via DAG and PKC, possibly by a store-independent mechanism. In addition in this cell type it appears that PKC and phosphorylation may play an important role in stimulating SOC activity in response to depletion of internal Ca²⁺ stores by CPA and BAPTA-AM.     

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl- sn -glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.     

Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control

The effects of porcine neuropeptide Y (NPY) regarding sympathetic vascular control were studied in vitro on isolated rat blood vessels. The 10(-9)M NPY enhanced (about two-fold) the contractile responses to transmural nerve stimulation (TNS), noradrenaline (NA) and adrenaline (about two-fold) in the femoral artery. Higher concentrations of NPY (greater than 10(-8)M) caused an adrenoceptor-resistant contraction per se. The TNS-evoked [3H]NA efflux was significantly reduced by NPY in a concentration-dependent manner (threshold 10(-9)M). The calcium antagonist, nifedipine, abolished the contractile effects of NPY and the NPY-induced enhancement of NA contractions but did not influence the prejunctional inhibition of [3H]NA release. Receptor-binding studies showed that the ratio of alpha 1-to alpha 2-adrenoceptors in the femoral artery was 30:1. The NPY did not cause any detectable change in the number of alpha 1-or alpha 2-adrenoceptor binding sites or in the affinity of alpha 2-binding sites, as revealed by prazosin- and clonidine-binding, respectively. The NPY also inhibited the TNS-evoked [3H]NA release (by 42-86%) in the superior mesenteric and basilar arteries and in femoral and portal veins. The NPY still depressed TNS-evoked [3H]NA secretion from the portal vein in the presence of phentolamine. The NPY caused a clear-cut contraction in the basilar artery, increased the contractile force of spontaneous contractions in the portal vein, while only weak responses were observed in the superior mesenteric artery and femoral vein. The NA-induced contraction was markedly enhanced by NPY in the superior mesenteric artery, only slightly enhanced in the portal vein and uninfluenced in the femoral vein. In conclusion, in all blood vessels tested, NPY depresses the TNS-evoked [3H]NA secretion via a nifedipine-resistant action. Furthermore, NPY exerts a variable, Ca2+-dependent vasoconstrictor effect and enhancement of NA and TNS contractions.     

The closing and opening of TRPC channels by Homer1 and STIM1

Influx of Ca(2+) is a central component of the receptor-evoked Ca(2+) signal. A ubiquitous form of Ca(2+) influx comes from Ca(2+) channels that are activated in response to depletion of the endoplasmic reticulum Ca(2+) stores and are thus named the store-operated Ca(2+) -influx channels (SOCs). One form of SOC is the transient receptor potential canonical (TRPC) channels. A major question in the field of Ca(2+) signalling is the molecular mechanism that regulates the opening and closing of these channels. All TRPC channels have a Homer-binding ligand and two conserved negative charges that interact with two terminal lysines of the stromal interacting molecule 1 (STIM1). The Homer and STIM1 sites are separated by only four amino acid residues. Based on available results, we propose a molecular mechanism by which Homer couples TRPC channels to IP(3) receptors (IP(3) Rs) to keep these channels in the closed state. Dissociation of the TRPCs-Homer-IP(3) Rs complex allows STIM1 access to the TRPC channels negative charges to gate open these channels.     

蛋白激酶C活性下调对钙库操纵的钙通道活性和气道平滑肌细胞增殖的作用

目的:研究蛋白激酶C(PKC)活性下调对大鼠气道平滑肌细胞(ASMCs)的Ca2+库操纵的Ca2+通道(SOC)和ASMCs增殖的影响。方法:分离培养大鼠支气管平滑肌细胞;利用激光共聚焦显微镜测定ASMCs的fluo-3/AM的荧光信号;利用长时间暴露于PKC活化剂诱导PKC活性下调,观察PKC活性下调对ASMCs的SOC通道和增殖的影响;Alamar blue还原率测定法测定ASMCs的增殖。结果:PKC激活剂PMA(10μmol/L)和PDBu(1μmol/L)作用24h下调PKC活性后可以抑制ASMCs的增殖,PKC抑制剂chelerythrine同样抑制ASMCs的增殖;PKC活性下调和chelerythrine均可以抑制ASMCs的SOC通道活性;小剂量PKC激活剂PMA(100nmol/L)可以促进ASMCs的增殖,这种作用被SOC通道抑制剂SKF-96365抑制。结论:PKC活性下调或抑制导致SOC通道的活性下降,表明PKC可能参与了SOC通道功能调控;SOC通道开放可能参与了PKC促进ASMCs增殖的作用。     

TRPC1/ORAI1复合体调节HUVECs SOC和ROC介导的Ca~(2+)内流和NO生成

目的研究瞬时受体电位通道1(TRPC1)/钙释放激活钙通道调节分子1(ORAI1)复合体在人脐静脉内皮细胞(HUVECs)钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)介导Ca~(2+)内流和NO生成中的作用。方法取2~3代HUVECs,将构建的TRPC1和ORAI1干扰质粒分别转染入HUVECs。用激光共聚焦显微镜观察细胞转染效果,real-time PCR和Western blot检测TRPC1、ORAI1 mRNA和蛋白的表达及抑制效率。细胞随机分组:特异性质粒转染组即实验组,未转染组即空白对照组及空质粒组(vehicle组),将上述3组细胞分别与CaR激动剂、ROC模拟剂TPA+CaR负性变构调节剂Calhex231、蛋白激酶C(PKC)抑制剂Ro31-8220、经典型PKCs和PKCμ抑制剂Go6967孵育,用荧光探针Fura-2/AM及DAF-FM负载方法同步检测[Ca~(2+)]i和NO。随后将构建的TRPC1和ORAI1干扰质粒同时转染入HUVEC,与CaR激动剂孵育后检测[Ca~(2+)]i和NO,用免疫共沉淀法检测TRPC1和ORAI1的相互作用。结果 1)与对照组相比,TRPC1及ORAI1组,mRNA和蛋白表达均明显降低(P0.05);2)在4种不同处理作用下,TRPC1及ORAI1转染组中细胞内Ca~(2+)浓度变化值和NO净荧光强度值均明显降低(P0.05);3)与对照组及单转染TRPC1及ORAI1组比,共转染组细胞内Ca~(2+)浓度变化值和NO净荧光强度值均明显降低(P0.05);4)TRPC1与ORAI1相互作用形成复合体,且在CaR激动剂的剌激下相互作用增强。结论 TRPC1与ORAI1复合体共同调节CaR经SOC和ROC激活介导的Ca~(2+)内流和NO生成。     

Protein kinase C suppresses spontaneous, transient, outwards K+ currents through modulation of the Na/Ca exchanger in guinea-pig gastric myocytes

The effect of protein kinase C (PKC) on the Ca2+-activated K+ current (IK,Ca) in guinea-pig gastric myocytes was studied using the whole-cell voltage-clamp technique. At a holding potential of 0 mV, IK,Ca, recorded as spontaneous, transient, outwards currents (STOCs), was markedly inhibited, both in mean amplitude (54 +/- 5%) and frequency (60 +/- 8%) by 1 microM phorbol 12, 13 dibutyrate (PDBu, n = 6). These effects were antagonized by pretreatment with 10 nM bisindolylmaleimide I (n = 5), a selective inhibitor of PKC. The possibility that the inhibition of STOCs was due to direct channel inhibition by PKC was addressed using inside-out or open-cell-attached patch-clamp techniques, the latter established using beta-escin. PDBu did not alter the conductance or open probability of the KCa channel in any mode, suggesting that PKC does not inhibit the KCa channel directly. To study the involvement of the Na/Ca exchanger in the inhibition of STOCs by PDBu, its operation was prevented by replacing Na+ in the internal solution by tris(hydroxymethyl)aminomethane (TRIS) and external Na+ by equimolar K+ and Ca2+-activated inwards K+ currents recorded at a holding potential of 0 mV. Neither the mean amplitude (96 +/- 8%) nor the frequency of these currents was inhibited significantly by 1 microM PDBu (n = 5). Like PDBu, 5 microM 2-(2-[4-(4-nitrobenzyloxy)phenyl]ethyl) isothiourea methanesulphonate (KB-R7943), a selective inhibitor of the reverse mode Na/Ca exchanger, also inhibited the mean amplitude (45 +/- 6%) and frequency (26 +/- 2%) of STOCs at the holding potential of 0 mV (n=6). The results suggest that the suppression of STOCs by PKC is mediated by inhibition of the Na/Ca exchanger.     

Contractile potentiation by endothelin-1 involves protein kinase C-delta activity in the porcine coronary artery.

The relationship between potentiation of serotonin (5-hydroxytryptamine, 5-HT)-induced contraction by endothelin-1 (ET-1) and the activity of protein kinase C (PKC) was examined in the porcine coronary artery. Low concentrations (30-100 pM) of ET-1 selectively potentiated the 5-HT-induced contraction 1.5 to 2 times over the control without any additional increase in myosin light-chain phosphorylation. The potentiation was attenuated by PKC down-regulation caused by phorbol 12-myristate 13-acetate. By Western blot analysis with isoform-specific antibodies to PKC, at least four isoforms (PKCalpha, PKCbeta1, PKCdelta and PKCzeta) were identified in the porcine coronary artery. PKCalpha and PKCdelta were mostly in the cytosol fraction, whereas PKCbeta1 and PKCzeta were almost equally distributed in the cytosol and membrane fractions in the resting and contractile states. Of the four isoforms, only PKCdelta was translocated from the cytosol to the membrane fraction during the contractile potentiation by ET-1. These results suggest that the activity of PKCdelta, a Ca2+-independent PKC isoform, is involved in the potentiation of 5-HT-induced contraction by ET-1 in the porcine coronary artery.     

Meal induced changes in hepatic and splanchnic circulation: a noninvasive Doppler study in normal humans

Summary The haemodynamic effects of a meal on the splanchnic and hepatic circulation were evaluated in 30 healthy volunteers, using Doppler ultrasonography. The resistance index (RI) of the superior mesenteric artery and of the left and right intrahepatic arteries, the portal vein blood flow as well as the ratio between maximal velocity in the left and right intrahepatic arteries and the adjacent portal vein were measured initially, then 15, 30, 45, and 60 min after the ingestion of a standard balanced liquid meal. Postprandial haemodynamic changes were maximal 30 min after the meal; at that time, mesenteric artery RI decreased significantly [mean –11% (SEM 14%)] whereas portal vein blood flow increased markedly [mean +79% (SEM 14%)]; a significant increase in hepatic artery RI was observed in both liver lobes. The ratio between maximal velocities of the intrahepatic artery and the intrahepatic portal vein was reduced significantly; this ratio decreased more markedly in the right lobe of the liver. These findings would suggest that there was an adaptation of hepatic artery to portal vein blood flow after a meal. The subsequent increase in intrahepatic portal vein flow velocity was found to be greater in the right lobe of the liver.     

Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.     

The effects of adrenaline and noradrenaline on venous return and regional blood flows in the anaesthetized cat with special reference to intestinal blood flow

1. In cats, a venous long-circuit technique was used to measure the blood flows in the superior vena cava and the hepatic, renal and iliac segments of the inferior vena cava. The sum of these flows gave the venous return (minus coronary and bronchial flows). In further experiments using an electromagnetic flowmeter, flow in the portal vein and in the superior mesenteric and coeliac arteries was measured.2. Approximately two-thirds of the hepatic blood flow is derived from the portal vein.3. After block of conduction in the cervical region of the spinal cord, the proportions of the venous return coming from each region during the control periods were not significantly altered although the arterial pressure and total venous return were decreased.4. Intravenous infusions of adrenaline caused an increase in venous return which was associated with a marked increase in hepatic blood flow. The increase in hepatic blood flow was due to an increase in flow in the superior mesenteric artery and portal vein. Flow in the coeliac artery remained unchanged. This response was unaffected by block of the cervical region of the spinal cord and by atropine or pentolinium.5. Intravenous infusions of noradrenaline caused little change in venous return or regional blood flows. Small increases in superior mesenteric artery flow were occasionally seen and on cessation of the infusion a large but brief increase occurred. These facts suggest that noradrenaline has a similar action to adrenaline but this is masked by concomitant vasoconstriction.     

Congenital absence of the portal vein in a middle-aged man

Congenital absence of the portal vein with systemic diversion of mesenteric blood is extremely rare. We report a case of a congenital absence of the portal vein, accidentally discovered in a 59-year-old man, completely asymptomatic and not associated with other malformations or biochemical disorders. Ultrasonography imaging showed the absence of the portal vein and the distal tract of superior mesenteric and splenic veins draining together into a dilated left renal vein. Computed tomography and magnetic resonance confirmed the presence of a congenital portosystemic venous shunt and also revealed two hepatic arteries: one arising from the celiac trunk and the other from the superior mesenteric artery.     

Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current ( I _cat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl- sn -glycerol (OAG) stimulates I _cat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on I _cat. With whole-cell recording of I _cat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked I _cat were much greater than those of OAG-induced I _cat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5) P ₃) in the pipette solution did not evoke I _cat but greatly potentiated the amplitude and rate of activation of OAG-induced I _cat. With isolated outside-out patches Ins(1,4,5) P ₃ markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5) P ₃ were mimicked by Ins(2,4,5) P ₃, 3-F-Ins(1,4,5) P ₃ and Ins(1,4) P ₂ but not by Ins(1,3,4,5) P ₄ and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of I _cat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.     

Age-related changes in adrenaline content of rat splanchnic blood vessels

The influence of ageing on the adrenaline content of the superior mesenteric artery and vein, renal artery and vein and portal vein was studied in 3-month- (young), 12-month- (adult) and 24-month-old (old) male Wistar rats using radioenzymatic assay for the measurement of catecholamine levels. Adrenaline concentrations were unchanged in the vascular wall of the blood vessels examined in adult rats, but were significantly decreased in the vascular wall of the superior mesenteric, renal and portal veins of old rats. In contrast, no age-dependent changes of adrenaline levels were found in the vascular wall of the superior mesenteric or renal arteries. The possibility that the loss of adrenaline concentrations in the venous vascular wall may be in some way related to the cardiovascular impairment occurring with age is discussed.     

The sites of action of cholecystokinin in decreasing meal size in pigs

The synthetic octapeptide of cholecystokinin (CCK-8) was infused at the rate of 67 ng/kg-minute into the jugular vein, carotid artery, portal vein, gastric branch of the splenic artery, and aortic artery both craniad and caudad to the origins of the cranial mesenteric and coeliac arteries of 39 young female pigs. Jugular, carotid and cranial erotic infusions resulted in significant reduction of meal size respectively to 65, 67 and 71% of control meal size (p less than 0.05). Gastric artery infusion resulted in a significant increase of meal size to 122% of control meal size (p less than 0.05). Portal vein and caudal aortic artery infusions had no significant effects on meal size. The results were interpreted as indicating a major site of action of CCK in reducing meal size in the bed of the cranial mesenteric or coeliac arteries, but excluding the stomach and liver.     

Intracellular Mg(2+) hyperpolarizes rabbit coronary artery smooth muscle cells by differential modulation of Ca(2+)(i)-dependent ion channels

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (> or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.     

Properties of the inward-rectifying Cl– channel in rat choroid plexus: regulation by intracellular messengers and inhibition by divalent cations

The properties of the inward-rectifying Cl- conductance in rat choroid plexus epithelial cells were investigated to allow comparisons to be made with ClC-2. All experiments were performed using the whole-cell configuration of the patch-clamp method. The conductance was transiently activated using an electrode solution which contained 375 nM catalytic subunit of protein kinase A (PKA). PKA failed to activate the conductance, however, when cells were pre-incubated with phorbol esters, which activate protein kinase C [1 microM phorbol 12-myristate 13-acetate (PMA) and 1 microM phorbol 12,13-dibutyrate (PDBu)]. Sustained activation of the conductance by PKA was observed in Ca2+-free conditions (5 mM BAPTA in the electrode solution), or when 100 nM calphostin C, a PKC inhibitor, was added to the electrode solution. The inward-rectifying Cl- conductance in choroid plexus is therefore similar to ClC-2 in that it is inhibited by PKC. The inward-rectifying conductance was blocked when Cd2+ (30 and 300 microM) and Zn2+ (1, 30 and 300 microM) were added to the bath solution. ClC-2 channels are also blocked by Zn2+ and Cd2+. The magnitude of the inward conductance was dependent on the concentration of ATP in the electrode solution. The conductance was not observed when ATP in the electrode was replaced with non-hydrolysable ATP analogues [adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) and 5'-adenylylimidodiphosphate (AMP-PNP)), but it was supported by UTP and GTP. These data contrast with those of previous studies in which ClC-2 channels were activated in the absence of ATP. In conclusion, the inward-rectifying Cl- channel in rat choroid plexus shares some properties with ClC-2 (inhibition by PKC and block by divalent cations), but differs in that it depends on intracellular ATP.     

The relationship of the inferior mesenteric vein with arteries

The investigation was based on the results of roentgen-anatomical study of 155 digestive tract complexes obtained from 155 cadavers of humans of both genders aged 17-90 years. It was established that in 91% (in 141 of 155) of the cases the trunk of the inferior mesenteric vein (IMV) was accompanied by the arteries only in the lower part (by inferior mesenteric artery) and in the middle part (by the left colic artery). The terminal part of IMV was separated from the artery. In 9% of (in 14 of 155) cases the trunk of the IMV is accompanied along the whole extent by different arteries (from down upwards): by inferior mesenteric artery and left colic artery from inferior mesenteric artery, and in its terminal part either by an additional anastomosis between the superior and inferior mesenteric arteries (in 5 of 14 cases), or by an additional anastomosis between superior and inferior mesenteric arteries and the branch or trunk of the proximal colic artery from the superior mesenteric artery (in 9 of 14 cases).     

KB-R7943 reveals possible involvement of Na(+)-Ca2+ exchanger in elevation of intracellular Ca2+ in rat carotid arterial myocytes.

A Na(+)/Ca(2+) exchanger (NCX) is one of the major regulators of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cardiac muscle cells. Although vascular smooth muscle myocytes also express NCX proteins, their functional role has not been clear, mainly due to the lack of specific inhibitors of NCX and relatively low levels of expression of NCX. In the present study, we have examined the involvement of NCX in the Na(+) deficient (0 Na(+)) elevation of [Ca(2+)](i) in rat carotid arterial myocytes using KB-R7943, an inhibitor of NCX. Perfusion with a Na(+)-free bathing solution, prepared by replacement of Na(+) with N-methyl-D-glucamine, induced an elevation of [Ca(2+)](i), which was effectively inhibited by KB-R7943 (IC(50)=3.5 microM). This inhibition was reversed by washout of KB-R7943. In contrast, D600, a blocker of voltage dependent L-type Ca(2+) channels (VDCC), did not affect the 0 Na(+)-induced elevation of [Ca(2+)](i). Treatment of myocytes with ryanodine abolished the elevation of [Ca(2+)](i) caused by caffeine but not that caused by 0 Na(+). Application of Cd(2+), which is known to block NCX as well as VDCC, also significantly inhibited the 0 Na(+) induced elevation. These results suggest that KB-R7943 inhibits the extracellular Na(+) dependent ([Na(+)](o)) change in [Ca(2+)](i) in rat carotid arterial myocytes, which is presumably activated by the reverse mode of NCX.