Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate

The cytosolic Ca(2+) concentration ([Ca(2+)](c)) controls diverse cellular events via various Ca(2+) signaling patterns; the latter are influenced by the method of cell activation. Here, in single-voltage clamped smooth muscle cells, sarcolemma depolarization generated uniform increases in [Ca(2+)](c) throughout the cell entirely by Ca(2+) influx. On the other hand, the Ca(2+) signal produced by InsP(3)-generating agonists was a propagated wave. Using localized uncaged InsP(3), the forward movement of the Ca(2+) wave arose from Ca(2+)-induced Ca(2+) release at the InsP(3) receptor (InsP(3)R) without ryanodine receptor involvement. The decline in [Ca(2+)](c) (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP(3)-mediated Ca(2+) release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP(3) receptors produced by an increased [Ca(2+)](c) rather than a reduced luminal [Ca(2+)] or an increased cytoplasmic [InsP(3)]. The deactivation of the InsP(3) receptor was delayed in onset, compared with the time of the rise in [Ca(2+)](c), persisted (>30 s) even when [Ca(2+)](c) had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca(2+) signaling patterns in smooth muscle. Sarcolemma Ca(2+) entry increases [Ca(2+)](c) uniformly; agonists activate InsP(3)R and produce Ca(2+) waves. Waves progress by Ca(2+)-induced Ca(2+) release at InsP(3)R, and persistent Ca(2+)-dependent inhibition of InsP(3)R accounts for the decline in [Ca(2+)](c) at the back of the wave.     

The Ca2+ release activities of D-myo-inositol 1,4,5-trisphosphate analogs are quantized

Comparison is made between several synthetic stereo and positional isomers of D-myo-inositol 1,4,5-trisphosphate (D-myo-1,4,5-IP3) with respect to their ability to mobilize calcium from the internal stores of saponin-permeabilized rat basophilic leukemia cells. D- and L-myo-Inositol 1,4,5-trisphosphates, D- and L-myo-inositol 2,4,5-trisphosphates, D- and L-chiro-inositol 1,3,4-trisphosphates, D,L-trans-1,2-cyclohexane-diol bisphosphate, D,L-myo-inositol 4,5-bisphosphate, L-glycerol 1,2-bisphosphate, glycerol 1,3-bisphosphate and D,L-(1R,3R,4R)-1-phosphoryloxymethyl-trans-3,4-cyclohexanediol bisphosphate were tested. The analogs, each of which contains a vicinal trans-1,2-diol-bisphosphate motif, displayed potencies that were distributed over a 10(4)-fold range of concentration and fell into 4 distinct classes of activity.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Molecular recognition of adenophostin, a very potent Ca2+ inducer, at the D-myo-inositol 1,4,5-trisphosphate receptor.

The recognition mode of adenophostin A at the D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor was investigated. Comparison of conformations of Ins(1,4,5)P(3) and adenophostin A by using the combination of NMR and molecular mechanics (MM) calculations demonstrated that adenophostin A adopted a moderately extended conformation regarding the distance between the 2'-phosphoryl group and the 3' ',4' '-bisphosphate motif, as suggested previously [Wilcox, R. A. et al. (1995) Mol. Pharmacol. 47, 1204-1211]. Based on the nuclear Overhauser effect (NOE) observed between 3'-H and 1' '-H and on MM calculations, the molecular shape of adenophostin A proved to be an extended form at least in solution, in contrast to Wilcox's compactly folded, preliminary hairpin model. GlcdR(2,3',4')P(3), an adenophostin analogue without adenine moiety, was found to be less potent than adenophostin A and almost equipotent to Ins(1,4,5)P(3). We propose the possibility that (i) the optimal spatial arrangement of the three phosphoryl groups and/or (ii) the interaction of the adenine moiety of adenophostin A with the putative binding site, if it exists in the vicinity of the Ins(1,4,5)P(3)-binding site, might account for the exceptional potency of adenophostin A.     

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels.

The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.     

The initial inositol 1,4,5-trisphosphate response induced by histamine is strongly amplified by Ca(2+) release from internal stores in smooth muscle

We have studied the Ca(2+)-dependence and wortmannin-sensitivity of the initial inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) response induced by activation of either histamine or muscarinic receptors in smooth muscle from guinea pig urinary bladder. Activation of H(1) receptors with histamine (100 microM) produced a significant elevation in Ins(1,4,5)P(3) levels with only 5s stimulation and in the presence of external Ca(2+). However, this response was abolished fully by either the prolonged absence of external Ca(2+) or the depletion of internal Ca(2+) stores with thapsigargin (100nM) or ryanodine (10 microM). In contrast, the same conditions only slightly reduced the initial Ins(1,4,5)P(3) response induced by carbachol. The prolonged incubation of smooth muscle in 10 microM wortmannin to inhibit type III PI 4-kinase abolished both the early histamine-evoked Ins(1,4,5)P(3) and Ca(2+) responses. Conversely, wortmannin did not alter Ca(2+) release induced by carbachol, despite a partial reduction of its Ins(1,4,5)P(3) response. Collectively, these data indicate that the detectable histamine-induced increase in Ins(1,4,5)P(3) is more the consequence of Ca(2+) release from internal stores than a direct activation of phospholipase C by H(1) receptors. In addition, the effect of wortmannin implies the existence of a Ca(2+)-dependent amplification loop for the histamine-induced Ins(1,4,5)P(3) response in smooth muscle.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Kinetics of ATP-dependent Ca2+ uptake by permeabilized rat enterocytes. Effects of inositol 1,4,5-trisphosphate

Isolated rat enterocytes were permeabilized by saponin treatment. 45Ca2+ was accumulated by these cells when provided with ATP in a medium containing Ca2+ ligands. The use of oxalate, vanadate and mitochondrial inhibitors indicated that both non-mitochondrial and mitochondrial pools are involved. Kinetic analysis of non-mitochondrial Ca2+ uptake revealed a Km of 0.1 microM Ca2+ and a Vmax of 0.4 nmol Ca2+/mg protein X min for this Ca2+-pumping ATPase activity. Mitochondria started to take up Ca2+ between 0.2 and 0.3 microM free Ca2+ reaching maximal rates around 2 microM. At 1 microM free Ca2+ mitochondria accumulated 20 times more Ca2+ than the non-mitochondrial pool. Inositol 1,4,5-trisphosphate released 40% of the Ca2+ content of the non-mitochondrial pool. Half-maximal release was observed at 0.5 and 1.5 microM IP3 in duodenal and ileal cells respectively. These findings support the possibility that the phosphatidyl inositide metabolism plays a role in regulation of electrolyte transport in enterocytes.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum and contraction in crustacean muscle

Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]: the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (greater than 10 microM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 less than 5 s) and then slowly returned (t1/2 less than 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] less than 100 microM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 microM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Tianeptine's effects on spontaneous and Ca2+-induced uterine smooth muscle contraction

Tianeptine is a novel anti-depressant with an efficacy equivalent to that of classical anti-depressants. Additional beneficial effects include neuroprotection, anti-stress and anti-ulcer properties whose molecular mechanisms are still not completely understood but may involve changes in the anti-oxidant defence system. Herein, we have studied the effects of tianeptine on both contractile activity of isolated rat uteri and components of the endogenous anti-oxidative defence system. Tianeptine-induced dose-dependent inhibition of both spontaneous and Ca2+-induced contraction of uterine smooth muscle. The effect was more pronounced in the latter. Tianeptine treatment increased glutathione peroxidase (GSH-Px) and catalase (CAT) activities in spontaneous and Ca2+-stimulated uteri. A significant decrease in glutathione-reductase (GR) activity in both spontaneous and Ca2+-induced uterine contractions after tianeptine treatment indicated a reduction in reduced glutathione and consequently a shift toward a more oxidised state in the treated uteri. In spontaneously contracting uteri, tianeptine caused a decrease in copper-zinc SOD (CuZnSOD) activity. Tianeptine's anti-depressant effects may be accomplished by triggering a cascade of cellular adaptations including inhibition of smooth muscle contractility and an adequate anti-oxidative protection response.     

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.     

Inositol 1,4,5-trisphosphate induced mobilization of Ca2+ from rat brain synaptosomes

Inositol 1,4,5-trisphosphate (IP₃) was found to release Ca²⁺ from presynaptic nerve endings (synaptosomes) made permeable with saponin. ATP-dependent Ca²⁺ uptake was carried out until equilibrium was reached. Addition of IP₃ produced a rapid release of Ca²⁺, which was complete within 60 sec, followed by Ca²⁺ reaccumulation to the original level in 5–7 min. Cholinergic receptor stimulation with muscarine also produced a similar Ca²⁺ release from synaptic endoplasmic reticulum. Ca²⁺ release by IP₃ was not detectable in the absence of the mitochondrial inhibitors oligomycin or sodium azide. Reaccumulation of Ca²⁺ was prevented by the presence of vanadate, a potent inhibitor of Ca²⁺/Mg²⁺ ATPase. Half maximal and near complete release of Ca²⁺ took place at 0.4 M and 3 M IP₃ concentrations, respectively. These studies demonstrate for the first time IP₃ mobilization of Ca²⁺ from endoplasmic reticulum within synaptic plasma membranes.