Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle

In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.     

Effects of semotiadil fumarate (SD-3211) and its enantiomer, SD-3212, on the changes in cytosolic Ca2+ and tension caused by KCl and norepinephrine in isolated rat aortas

Semotiadil fumarate (SD-3211), a Ca2+ channel blocker of benzothiazine derivative and its (S)-(-)-enantiomer (SD-3212), inhibited K(+)- and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K(+)-contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD-3212 (10 microM) inhibited the increase in cytosolic Ca2+ ([Ca2+]i) induced by 65.4 mM K+ in fura-2-loaded preparations as well as diltiazem and bepridil (10 microM). On the other hand, semotiadil and SD-3212 (10 microM) inhibited only the early phase of increase in [Ca2+]i induced by 1 microM NE. After 5 min, no significant effect on [Ca2+]i was observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 microM affected neither the increase in [Ca2+]i nor the contraction induced by NE. Semotiadil and SD-3212 inhibited the transient contraction induced by 1 microM NE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD-3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+ channels but also through other mechanisms, such as inhibition of Ca2+ release from Ca2+ stores or decrease in sensitivity of the contractile elements to Ca2+.     

JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Ca2+ transient, cell volume, and microviscosity of the plasma membrane in smooth muscle

Despite pronounced differences by which membrane-depolarizing or phospholipase C-activating stimuli initiate contractile responses, a rise in [Ca2+]i is considered the primary mechanism for induction of smooth muscle contractions. Subsequent to the formation of the well-characterized Ca(2+)4-calmodulin complex, interaction with the catalytic subunit of myosin light chain kinase triggers phosphorylation of 20 kDa myosin light chain and activates actin-dependent Mg2+-ATPase activity, which ultimately leads to the development of tension. The present article reviews the fundamental mechanisms leading to an increase in [Ca2+]i and discusses the biochemical processes involved in the transient and sustained phases of contraction. Moreover, the commentary summarizes current knowledge on the modulatory effect of changes in the microviscosity of the plasma membrane on the Ca2+ transient as well as the contractile response of smooth muscle. Evidence has accumulated that these changes in microviscosity alter the activity of membrane-bound enzymes and affect the generation of endogenous mediators responsible for the regulation of cytosolic Ca2+ concentrations and for the [Ca2+]i-sensitivity of myosin light chain phosphorylation.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids

BACKGROUND: Recombinant human growth hormone (GH) improves in vivo cardiac function in rats with postinfarction heart failure (MI). We examined the effects of growth hormone (14 days of 3.5 mg. kg-1. d-1 begun 4 weeks after MI) on contractile reserve in left ventricular myocytes from rats with chronic postinfarction heart failure. METHODS AND RESULTS: Cell shortening and [Ca2+]i were measured with the indicator fluo 3 in myocytes from MI, MI+GH, control, and normal animals treated with GH (C+GH) under stimulation at 0.5 Hz at 37 degrees C. Cell length was similar in MI and MI+GH rats (150+/-5 and 157+/-5 microm) and was greater in these groups than in the control and C+GH groups (140+/-4 and 139+/-4 microm, P<0.05). At baseline perfusate calcium of 1.2 mmol/L, myocyte fractional shortening and [Ca2+]i transients were similar among the 4 groups. We then assessed contractile reserve by measuring the increase in myocyte fractional shortening in the presence of high-perfusate calcium of 3.5 mmol/L. In the control and C+GH groups, myocyte fractional shortening and peak systolic [Ca2+]i were similarly increased in the presence of high-perfusate calcium. In the presence of high-perfusate calcium, both myocyte fractional shortening and peak systolic [Ca2+]i were depressed in the MI compared with the control groups. In contrast, myocyte fractional shortening (14.1+/-.9% versus 11.1+/-.9%, P<0.05) and peak systolic [Ca2+]i (647+/-43 versus 509+/-37 nmol/L, P<0.05) were significantly higher in MI+GH than in MI rats and were comparable to controls. Left ventricular myocyte expression of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA-2) and left ventricular SERCA-2 protein levels were increased in MI+GH compared with MI rats. CONCLUSIONS: Calcium-dependent contractile reserve is depressed in myocytes from rats with postinfarction heart failure. Long-term growth hormone therapy increases contractile reserve by restoring normal augmentation of systolic [Ca2+]i in myocytes from rats with postinfarction heart failure.     

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca2+ in rat aorta

We investigated the effects of palmitoyl-L-alpha-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K+, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective alpha2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10(-6) M to 10(-4) M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K+ - and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10(-5) M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10(-5) M) markedly augmented the increase in both cytosolic free Ca2+ ([Ca2+]i) and contractile tension induced by UK14,304, high K+ and PMA. Nicardipine (10(-7) M) and 10(-6) M Ro-31-8220 (?1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-++ +indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca2+]i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K+ -induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca2+]i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels

BACKGROUND: A reduction in oxygen tension in the lungs is believed to inhibit a voltage-dependent K+ (Kv) current, which is thought to result in membrane depolarization leading to hypoxic pulmonary vasoconstriction (HPV). However, the direct mechanism by which hypoxia inhibits Kv current is not understood. METHODS AND RESULTS: Experiments were performed on rat pulmonary artery resistance vessels and single smooth muscle cells isolated from these vessels to examine the role of Ca2+ release from intracellular stores in initiating HPV. In contractile experiments, hypoxic challenge of endothelium-denuded rat pulmonary artery resistance vessels caused either a sustained or transient contraction in Ca2+-containing or Ca2+-free solution, respectively (n=44 vessels from 11 animals). When the ring segments were treated with either thapsigargin (5 micromol/L), ryanodine (5 micromol/L), or cyclopiazonic acid (5 micromol/L) in Ca2+-containing or Ca2+-free solution, a significant increase in pulmonary arterial tone was observed (n=44 vessels from 11 animals). Subsequent hypoxic challenge in the presence of each agent produced no further increase in tone (n=44 vessels from 11 animals). In isolated pulmonary resistance artery cells loaded with fura 2, hypoxic challenge, thapsigargin, ryanodine, and cyclopiazonic acid resulted in a significant increase in [Ca2+]i (n=18 cells from 6 animals) and depolarization of the resting membrane potential (n=22 cells from 6 animals). However, with prior application of thapsigargin, ryanodine, or cyclopiazonic acid, a hypoxic challenge produced no further change in [Ca2+]i (n=18 from 6 animals) or membrane potential (n=22 from 6 animals). Finally, application of an anti-Kv1.5 antibody increased [Ca2+]i and caused membrane depolarization. Subsequent hypoxic challenge resulted in a further increase in [Ca2+]i with no effect on membrane potential (n=16 cells from 4 animals). CONCLUSIONS: In rat pulmonary artery resistance vessels, an initial event in HPV is a release of Ca2+ from intracellular stores. This rise in [Ca2+]i causes inhibition of voltage-dependent K+ channels (possibly Kv1.5), membrane depolarization, and an increase in pulmonary artery tone.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

Contraction and intracellular calcium ([Ca2+]i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22-24 degreesC stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca2+]i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca2+]i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca2+.     

Polarity of epithelial cells of the liver. Cellular and molecular mechanisms, and pathologic changes

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.     

The Rho GTPases in macrophage motility and chemotaxis

The present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin- in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imida zole . HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 microM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.     

Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca2+]i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca2+]i transient was reduced following treatment with 100 nM phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5 microM) and calphostin C (10 microM) increased [Ca2+]i transient amplitude, elevated basal [Ca2+]i and slowed the decay of the [Ca2+]i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca2+ channels was monitored indirectly as the rate of Mn2+ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn2+ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca2+ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca2+ content may be secondary to alterations of sarcolemmal Ca2+ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca2+ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca2+]i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca2+ influx, which may play a key role in setting the basal tone of cardiac muscle.     

Dietary and physiological studies to investigate the relationship between calcium and magnesium signalling in the mammalian myocardium

This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca2+) and magnesium (Mg2+) signalling in the mammalian myocardium. Rats maintained on a low Mg2+ diet (LMD; 39 mg Kg-1 Mg2+ in food) consumed less food and grew more slowly than control rats fed on a control Mg2+ diet (CMD; 500 mg Kg-1 Mg2+ in food). The Mg2+ contents of the heart and plasma were 85 +/- 3% and 34 +/- 6.5%, respectively relative to the control group. In contrast, Ca2+ contents in the heart and plasma were 177 +/- 5% and 95 +/- 3%. The levels of potassium (K+) was raised in the plasma (129 +/- 16%) and slightly decreased in the heart (88 +/- 6%) compared to CMD. Similarly, sodium (Na+) contents were slightly higher in the heart and lowered in the plasma of low Mg2+ diet rats compared to control Mg2+ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg2+]o resulted in a small transient increase in the amplitude of contraction compared to control [Mg2+]o (1.2 mM). In contrast, elevated [Mg2+]o (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca2+ current (ICa,L) was significantly (p < 0.001) attenuated in cells dialysed with 7.1 mM Mg2+ compared to cells dialysed with 2.9 microM Mg2+. The results indicate that hypomagnesemia is associated with decreased levels of Mg2+ and elevated levels of Ca2+ in the heart and moreover, internal Mg2+ is able to modulate the Ca2+ current through the L-type Ca2+ channel which in turn may be involved with the regulation of contractile force in the heart.     

Enhancement of vascular action of arginine vasopressin by diminished extracellular sodium concentration

The present study was undertaken to examine the effects of diminished extracellular sodium concentration on the vascular action of arginine vasopressin (AVP) in cultured rat vascular smooth muscle cells (VSMC). The preincubation of cells with the 110 mM extracellular Na+ ([Na+]e) solution supplemented with 30 mM choline chloride for 60 minutes enhanced the effect of AVP- (1 x 10(-8) M) induced VSMC contraction. The treatment of 110 mM [Na+]e solution also enhanced the cellular contractile response to the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetyl-glycerol. Furthermore, preincubation with the 110 mM [Na+]e solution also potentiated the effect of 1 x 10(-8) M AVP, but not 1 x 10(-6) M, to increase the cytosolic-free Ca2+ ([Ca2+]i) concentration. The 110 mM [Na+]e media decreased the basal intracellular Na+ concentration and increased intracellular 45Ca2+ accumulation, basal [Ca2+]i and AVP-produced 45Ca2+ efflux. These effects of 110 mM [Na+]e solution to enhance the vascular action of AVP were abolished by using Ca(2+)-free 110 mM [Na+]e solution during the preincubation period. The preincubation with the 110 mM [Na+]e solution did not change either the Kd and Bmax of AVP V1 receptor of VSMC or the AVP-induced production of inositol 1,4,5-trisphosphate. The present in vitro results therefore indicate that the diminished extracellular fluid sodium concentration within a range observed in clinical hyponatremic states enhances the vascular action of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phorbol ester, 12-deoxyphorbol 13-isobutylate (DPB), on muscle tension and cytosolic Ca2+ in rat anococcygeus muscle

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.