Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells.

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [3H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1-10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100-200 microM) and the NO synthase inhibitor L-NAME (100 microM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Spontaneous activity and stretch-induced contractile differentiation are reduced in vascular smooth muscle of miR-143/145 knockout mice.

AIM: Stretch is essential for maintaining the contractile phenotype of vascular smooth muscle cells, and small non-coding microRNAs are known to be important in this process. Using a Dicer knockout model, we have previously reported that microRNAs are essential for stretch-induced differentiation and regulation of L-type calcium channel expression. The aim of this study was to investigate the importance of the smooth muscle-enriched miR-143/145 microRNA cluster for stretch-induced differentiation of the portal vein. METHODS: Contractile force and depolarization-induced calcium influx were determined in portal veins from wild-type and miR-143/145 knockout mice. Stretch-induced contractile differentiation was investigated by determination of mRNA expression following organ culture for 24 h under longitudinal load by a hanging weight. RESULTS: In the absence of miR-143/145, stretch-induced mRNA expression of contractile markers in the portal vein was reduced. This was associated with decreased amplitude of spontaneous activity and depolarization-induced contractile and intracellular calcium responses, while contractile responses to 5-HT were largely maintained. We found that these effects correlated with a reduced basal expression of the pore-forming subunit of L-type calcium channels and an increased expression of CaMKIIδ and the transcriptional repressor DREAM. CONCLUSION: Our results suggest that the microRNA-143/145 cluster plays a role in maintaining stretch-induced contractile differentiation and calcium signalling in the portal vein. This may have important implications for the use of these microRNAs as therapeutic targets in vascular disease.

Nitric oxide relaxes rat tail artery smooth muscle by cyclic GMP-independent decrease in calcium sensitivity of myofilaments.

The effects of authentic nitric oxide (NO, 10(-6) M) and NO-donors such as sodium nitroprusside (SNP, 10(-5) M) and glyceryl trinitrate (GTN, 10(-4) M) on contractile force and free intracellular calcium level ([Ca2+]i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca2+ was measured using chemically permeabilized (alpha-toxin, beta-escin, Triton X-100) vascular rings. [Ca2+]i and contractile activity were measured simultaneously. The relationship of [Ca2+]i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca2+]i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10(-5) M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10(-6) M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO- and SNP-induced reduction in [Ca2+]i. On the contrary, GTN induced neither relaxation nor decrease in [Ca2+]i on application of both LY83583 and ODQ. Tail artery rings permeabilized with alpha-toxin, beta-escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5 x 10(-3) M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10(-5) M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of beta-escin permeabilized rings in low Ca2+ (10(-8) M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca2+]i and may involve activation of protein phosphatase(s).

Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca(2+) handling.

The roles of intracellular Ca(2+) stores and ryanodine (Ry) receptors for vascular Ca(2+) homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 - 100 microM) for up to 4 days. Acute exposure to Ry or the non-deactivating ryanodine analogue C(10)-O(eq) glycyl ryanodine (10 microM) eliminated Ca(2+) release responses to caffeine (20 mM) and noradrenaline (NA, 10 microM), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to RY: Ry receptor protein was detected on Western blots in arteries cultured either with or without RY: Brief Ca(2+) release events (sparks) were absent after culture with Ry, whereas Ca(2+) waves still occurred. The propagation velocity of waves was equal ( approximately 19 microm s(-1)) in tissue cultured either with or without RY: Inhibition of Ca(2+) accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 microM) decreased contractility due to Ca(2+)-induced cell damage. In contrast, culture with Ry did not affect contractility. Removal of Ca(2+) from the cytosol following a Ca(2+) load was retarded after Ry culture. Thapsigargin reduced the rate of Ca(2+) removal in control cultured rings, but had no effect after Ry culture. It is concluded that intracellular Ca(2+) stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca(2+) sparks and for SR-dependent recovery from a Ca(2+) load, but not for Ca(2+) waves or basal Ca(2+) homeostasis.

Role of vasoactive intestinal polypeptide (VIP) in the neurogenic vasodilatation of the portal vein in the rabbit.

A coarse network of nerve fibres displaying immunoreactivity for vasoactive intestinal polypeptide (VIP) was found in the wall of the hepatic portal vein of the rabbit. Electrical field stimulation of the rabbit portal vein in vitro, in the presence of adrenergic and cholinergic blockade, caused a marked relaxation of the vessel and a release of VIP into the perfusate. Addition of VIP to the tissue bath elicited a concentration-dependent inhibition of the mechanical activity of the portal vein. The results suggest that VIP containing neurones might participate in the non-cholinergic, non-adrenergic vasodilatation of the portal vein in the rabbit.

Phosphorus-31 NMR studies of smooth muscle from guinea-pig taenia coli.

Phosphorus-31 NMR spectra of superfused isometrically mounted guinea-pig taenia coli were obtained using a horizontal probe at 103.2 MHz. The spectra showed resonances for ATP, phosphocreatine (PCr), and a sugar phosphate resonance. The PCr/ATP ratio was between 1.5 and 2.0 consistent with chemical analysis of tissue extracts. The level of PCr, but not of ATP, decreases reversibly during contraction or inhibition of respiration. These conditions did not cause substantial changes in the intracellular pH, which was 7.0 +/- 0.1.

The diagnosis of carpal tunnel syndrome. Sensitivity and specificity of some clinical and electrophysiological tests.

The study group consisted of 100 persons referred with suspected carpal tunnel syndrome. Clinical and neurophysiological examinations were performed blinded from each other. The gold standard for the carpal tunnel syndrome (CTS) diagnosis was based on the results of these examinations but relief of CTS symptoms after surgery was also required. The sensitivity and specificity for the combined results of the clinical examinations were 94% and 80% respectively, and for the neurophysiological examinations, 85% and 87%. Of the neurophysiological methods used, the quotient of sensory nerve conduction velocity between palm to wrist and wrist to elbow was best and the cut-off for this test was studied by means of an ROC-curve. According to our results clinical examination by an experienced doctor seems to be sufficient if there are typical symptoms of carpal tunnel syndrome, but if there is a history of pain, atypical symptoms or earlier fractures in the arm, wrist or hand, it is important to add a neurophysiological examination.

Injuries caused by firewood splitting machines.

The aim of this paper is to present the types of injury caused by firewood splitting machines and also to elucidate the accident mechanism. The study is based on 15 cases. The machine has a rotating spiral cone, and usually the victims' gloved fingertips were caught by the point of the cone. This led to either amputations, usually of radial fingers and/or penetrating wounds through the middle of the hand. In most cases the accidents could not be blamed on bad working techniques. The study of the mechanisms of injury points to insufficient protective devices in a machine construction which has a potentially dangerous working principle.

Oats (Avena sativa) reduce atherogenesis in LDL-receptor-deficient mice.

AIM: The cholesterol-lowering properties of oats, largely ascribed to its contents of soluble fibers, beta-glucans, are well established, whereas effects on atherogenesis are less well elucidated. Oats also contains components with reported antioxidant and anti-inflammatory effects that may affect atherogenesis. In this work we examined effects of oat bran on plasma cholesterol, markers of inflammation, eNOS expression and development of atherosclerosis in LDL-receptor-deficient (LDLr(-/-)) mice. METHODS AND RESULTS: Female LDLr(-/-) mice were fed Western diet+/-oat bran. Two concentrations of oat bran (40 and 27%) were compared regarding effects on plasma lipids. There was a dose-dependent reduction of plasma cholesterol by 42 and 20% with 40 and 27% oat bran, respectively. Both concentrations also lowered plasma triglycerides (by 45 and 33%) and relative levels of plasma LDL+VLDL. The reduction of plasma lipids was accompanied by increased faecal excretion of cholesterol and bile acids. Oat bran (40%) efficiently reduced atherosclerotic lesion area in the descending aorta (-77%) and aortic root (-33%). Plasma levels of fibrinogen and soluble vascular cell adhesion molecule-1 (VCAM-1) were significantly lower, and immunofluorescence of aortic sections revealed a 75% lower expression of VCAM-1 in oat-fed mice. The expression of eNOS protein in the aortic wall was increased in mice fed oat bran. CONCLUSIONS: Oat bran supplemented to a Western diet lowers plasma cholesterol, reduces levels of some inflammatory markers, increases eNOS expression and inhibits atherosclerotic lesion development in LDLr(-/-) mice. It remains to be investigated which components in oats contribute to these effects.

Cross-bridge kinetics and shortening in smooth muscle.

Stiffness measurements were performed on smooth muscle preparations from guinea-pig taenia coli to obtain information on the number of attached cross bridges under varying contractile conditions. The normalized stiffness of the cross-bridge system in smooth muscle may be of a magnitude similar to that assumed in skeletal muscle. Transition from isometric contraction to unloaded shortening was associated with a decrease in stiffness to 50% or less of the isometric value, slightly higher than that found in skeletal muscle fibers. Comparison of phasic (5 s) and tonic (5 min) contractions showed lower Vmax, intracellular [Ca2+], and myosin 20 kDa light chain phosphorylation at 5 min, indicating development of a latch state. Isometric force and stiffness were identical in the two types of contraction. However, stiffness during unloaded shortening was greater in the latch state, which may be the result of the presence of a population of cross bridges with a low rate constant for detachment.

Long-term effects of intracellular calcium and growth factors on excitation and contraction in smooth muscle.

Modulation of vascular smooth muscle cells from a contractile to a synthetic phenotype is thought to be important in the development of the atherosclerotic lesion. Such modulation depends on growth factors and is influenced by cell-cell and cell-matrix interactions. Whereas smooth muscle cells in the vessel wall are contractile, dispersed cells in culture rapidly modulate to synthetic phenotype, which complicates long-term in vitro studies. In contrast, vascular segments or smooth muscle strips in organ culture can maintain contractility for at least a week, sufficient for studies involving altered metabolism or protein expression. Examples are effects of endogenous polyamines on membrane ion channels and excitation-contraction coupling. While smooth muscle tissue is well preserved in serum-free culture, growth stimulation with fetal calf serum (FCS) causes multiple effects, including decreased contractility, ultrastructural changes, decreased expression of L-type Ca2+ channels, and increased SR release of Ca2+ via ryanodine receptors. These are all consequences of increased basal [Ca2+]i caused by FCS, as they are reversed by culture with verapamil in a concentration (1 microM) that does not inhibit stimulation of DNA and protein synthesis by FCS. The effects of FCS on contractility and Ca2+ channel expression are mimicked in serum-free culture with increased [Ca2+]i. Contractile protein patterns, including myosin isoform composition, are unaffected by FCS, suggesting that reversal to synthetic phenotype is limited and not the immediate cause of decreased contractility.

Oxygen consumption and lactate production of the rat portal vein in relation to its contractile activity.

Energy turnover in the isolated rat portal vein was investigated by measurement of oxygen consumption (JO2) and lactate production (JLA) under simultaneous recording of mechanical activity. In spontaneous activity under aerobic conditions and at optimal muscle length JO2 and JLA were 0.55 and 0.62 micromol/min X g, respectively, corresponding to an ATP-production of 4.3 micromol/min X G. When muscle length was changed, an approximately linear relation was found between energy turnover and mean isometric tension. The tension-indpendent part of ATP-production was 3.0 micromol/min X g. In Ca2+-free solution the metabolic rate was 20% lower still. JO2 was nearly equal in isometric contractions and in afterloaded isotonic contractions from the same initial muscle length. During a maximal tonic contracture in 5+-depolarized portal vein JO2 increased to about twice that in spontaneous activity. Changes in contracture force by variations in muscle length or in [Ca2+]0 were associated with identical linear relations between JO2 and active tension. This relation was less steep than the corresponding relation for spontaneous activity. The anaerobic lactate production of the portal vein was 2.7 times theaerobic leve. The accelerated glycolysis did not compensate for eliminated oxidative metabolism. Under substrate-free aerobic conditions no lactate was produced by the muscle and compared to the control situation JO2 declined more than could be accounted for by reduced mechanical activity alone. The metabolic turnover rate in relation to isometric tension is high in the rat portal vein compared to that of tonic vascular smooth muscle from larger vessels. This correlates with differences in dyanmic mechanical properties. At comaparable tension levels in the portal vein, the rate of cross-bridge turnover may be higher in spontaneous phasic activity than in sustained contracture.

Cross-bridge kinetics during shortening in early and sustained contraction of intestinal smooth muscle.

Mechanisms responsible for the decrease in shortening velocity after prolonged contraction ("latch" state) were investigated at identical force during early (20 s, "phasic") and sustained (5 min, "tonic") phases of high-K+ (25-30 mM) contractions in smooth muscle of guinea pig taenia coli. Cytoplasmic Ca2+ concentration, myosin light-chain phosphorylation, and maximum shortening velocity all declined from 20 s to 5 min of contraction. The time course of shortening following isotonic quick release was biexponential, with a fastest rate constant of approximately 80 s-1 in both phasic and tonic contractions. Stiffness was identical in phasic and tonic contraction; however, after a release to slack length and unloaded shortening, stiffness during restretch was greater in tonic contraction (51 vs. 43% of isometric stiffness after 16 ms of unloaded shortening). Stiffness decreased after release with a rate constant of approximately 200 s-1, slightly greater in phasic than in tonic contraction. The results indicate that the number of attached cross bridges during unloaded shortening, while substantially reduced relative to the isometric value, is higher in latch than in nonlatch, consistent with a lower detachment relative to attachment rate.

Force response to rapid length change during contraction and rigor in skinned smooth muscle of guinea-pig taenia coli.

1. Mechanical transients in fibre bundles of skinned smooth muscle of guinea-pig taenia coli at 21-22 degrees C were investigated by recording tension responses to length changes of up to 9%, complete within 0.3 ms. 2. The length-force relationship, recorded continuously during rapid stretch of a Ca(2+)-activated contracted muscle, was linear up to at least 2.5 times the isometric force, corresponding to a stretch of about 1%. The slope of the relationship (stiffness) increased with the velocity of stretch. 3. During rapid release (about 120 muscle lengths s-1) the length-force relationship was linear down to about 50% of the initial isometric force, reached at about 80 microseconds after the beginning of the release. At lower force the length-force relationship was concave upwards. The linear portion extrapolated to zero force at about -0.008 muscle lengths. In large releases the length-force plot approached the force baseline under an acute angle, and negative force was transiently exerted. 4. When the muscle was stretched back to the initial length after a shortening step, force transiently rose above the isometric force, but decayed back within a few milliseconds. Stiffness at the time of restretch was compared with that in the initial shortening step by plotting force vs. length, and was found to be decreased to 63% within 0.3 ms of a step to zero force. Stiffness decreased further with time at zero force, and after 256 ms was about 29% of the isometric value. 5. In rigor, caused by the introduction of ATP-free solution during the plateau of isometric contraction, fibre tension decreased to about 30% of the active tension, whereas stiffness relative to force increased; 82% of the initial stiffness in rigor was detected in a restretch immediately after a shortening step, decreasing to 59% at 256 ms. When the fibre was activated at suboptimal [Ca2+] to cause the same force as in rigor, stiffness was lower than in rigor and decreased more after a release. 6. After completion of a release-stretch cycle, stiffness was rapidly restored to the same value as in isometric contraction. Test stretches at different points in time after completion of the cycle revealed that most of the stiffness had been restored within 1 ms of the restretch, occurring concomitantly with a decay in force.(ABSTRACT TRUNCATED AT 400 WORDS)

Heat production in chemically skinned smooth muscle of guinea-pig taenia coli.

1. The rate of heat production of chemically skinned guinea-pig taenia coli smooth muscle at 25 degrees C was measured using microcalorimetric techniques. 2. Muscle strips were mounted isometrically and incubated in solutions containing MgATP (3.2 mM) and phosphocreatine (PCr, 12 mM), pH 6.9. Activation was obtained by the injection of Ca2+ into the sample compartment of the calorimeter. 3. The heat production rate of the resting preparation (pCa 9) was 0.40 +/- 0.03 mW g-1 wet weight (n = 23). During maximal activation (pCa 4.8) the heat rate increased to 1.12 +/- 0.07 mW g-1 (mean +/- S.E.M., n = 15). With stepwise increase in [Ca2+] from pCa 9 to 4.8 the energetic cost of force maintenance tended to increase at higher [Ca2+]. 4. After activation by Ca2+, the heat production rate reached its maximum while force was still increasing. 5. Changing ionic strength from 90 to 150 mM had no effect on either basal or activated heat rate. Oligomycin, amphotericin B and the adenylate kinase inhibitor Ap5A had no effect on the basal heat rate. 6. Exchanging ATP in the incubation medium for inosine triphosphate (ITP) reduced the force and heat production after injection of Ca2+. The basal heat production was not lowered when ATP was exchanged for ITP. 7. The observed enthalpy change for PCr splitting at 25 degrees C (pH 6.9, ionic strength 90 mM) was -28 +/- 3 kJ mol-1 (mean +/- S.E.M., n = 9). After correction for the phosphate equilibrium, buffer reactions, and Mg2+ binding to PCr and HPO42-, the net enthalpy change is calculated to be -39 +/- 3 kJ mol-1. 8. Heat production in the skinned smooth muscle consists of one basal component present in relaxed muscle, and one component associated with contraction. The nature of the basal heat production is unclear but does not seem to involve turnover of phosphate on the myosin light chains. The increase in the energetic tension cost with increasing activation by Ca2+ has implications for the understanding of the contractile mechanism in smooth muscle.

Functional role of aerobic glycolysis in rat portal vein.

The functional role of aerobic lactate production in the rat portal vein was investigated. Changing substrate from glucose (11.5 mM) to pyruvate (11.5 mM) or beta-hydroxybutyrate (3 mM) had virtually no effect on spontaneous mechanical activity. Lactate production (FLA) was smaller with pyruvate than with glucose (0.05 +/- 0.01 vs. 0.14 +/- 0.03 mumol g-1 min-1, n = 4). Addition of 0.5 mM iodoacetate to inhibit glycolysis abolished mechanical activity in 15-20 min with glucose as substrate, whereas with pyruvate the mechanical activity was only moderately reduced over this time period. With beta-hydroxybutyrate (3 mM) as substrate no aerobic lactate production was detected during normal spontaneous activity. Inhibition of cellular respiration with increasing concentrations of cyanide in beta-hydroxybutyrate medium led to a graded decrease in mechanical activity and FO2, but only a marginal increase in lactate production. With glucose as substrate, repeated stimulation with a combination of isoproterenol (10(-5) M) and papaverine (10(-4) M) gave similar increases in lactate production at each exposure. With beta-hydroxybutyrate some lactate production was found at the first stimulation, but decreased to be abolished at the third stimulation. The mechanical inhibition caused by the stimulation was however similar at the three exposures for both substrates. Lactate production induced by cAMP-raising stimulation in beta-hydroxybutyrate could be accounted for by glycogenolysis. These results show that aerobic glycolysis leading to net lactate production is not necessary for normal spontaneous mechanical activity or the relaxing effect of hypoxia or cAMP raising stimuli in rat portal vein.

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle.

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca2+ channels. Spermine and spermidine (0.1-1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 microM), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 microM). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca2+ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca2+ current responsible for generation of action potentials.

Contractures induced by reversed Na+/Ca2+ exchange in rat portal vein: effects of calcium antagonists.

Spontaneous electrical and mechanical activity was abolished in isolated preparations of rat portal vein by exposure to K+-free Krebs solution. This procedure probably also increased the intracellular [Na+] owing to interference with the active transmembrane Na+-K+ transport. Contractures could be induced under these conditions by lowering extracellular [Na+] from the control level of 144 to 17 mM using sucrose, TrisCl, or LiCl as NaCl substitutes. Contractile force depended on the type of substitute: sucrose greater than TrisCl greater than LiCl. These contractures are thought to be caused by influx of extracellular Ca2+ through the Na+/Ca2+ exchanger, which otherwise transports Ca2+ in the opposite direction at normal transmembrane Na+ gradient. The contractile response to low Na+ was rapidly and completely abolished in nominally Ca2+-free medium; it was strongly inhibited by 0.4 mM MnCl2 but was not affected by high concentrations of the organic calcium antagonists, felodipine (10(-6) M), verapamil (10(-5) M), or diltiazem (10(-5) M). We conclude that the Na+/Ca2+-exchanger is an effective pathway for Ca2+ transport over vascular smooth muscle cell membrane; this pathway is not blocked by calcium antagonists.