Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Ca2+-dependent actin remodeling in the contracting A7r5 cell

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the -actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca²⁺]i by A₂₃₁₈₇ and thapsigargin on - and -actin remodeling. The effects of A₂₃₁₈₇ and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the - and -actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, -actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca²⁺]i resulted in partial or complete dissolution of -actin cables without further remodeling. In addition, PDBu-mediated -actin remodeling was blocked in the presence of A₂₃₁₈₇. Increased [Ca²⁺]i also caused dispersal of -actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for -actin cytoskeletal structure. The incubation of cells in calcium-free media prevented -actin dissolution by A₂₃₁₈₇/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of -actin cables by increased [Ca²⁺]i. The results suggest that the sustained elevation of [Ca²⁺]i beyond a threshold level initiates depolymerization of -actin but not -actin. It further appears that PDBu-induced -actin remodeling requires Ca²⁺ but increases of [Ca²⁺]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits -actin dissolution in the presence of A₂₃₁₈₇/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing -actin structure in the activated cell.     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

ATP-induced intracellular Ca2+ signals in isolated human insulin-secreting cells

Using isolated -cells from human islets of Langerhans we have demonstrated that purinergic receptor agonists are functionally coupled to rises in the intracellular calcium ion concentration ([Ca²⁺]_i). The effects of ATP, ADP and AMP have been examined over a range of concentrations, 0.5 to 500M. The actions of ATP were more potent than those of either ADP or AMP suggesting that a P₂-type of purinergic receptor operates in these cells. Responses to ATP were concentration-related, but exhibited marked desensitisation at high concentrations (>100M). Purinergic receptor agonists elevate [Ca²⁺]_i by mechanisms that involve both Ca²⁺ influx and Ca²⁺ mobilisation from intracellular stores. The physiological significance of our data has been discussed, and related to previous studies carried out upon rodent and clonal insulin-secreting cells.     

Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein

Enzymatically dispersed smooth muscle cells of the guinea-pig portal vein were studied by the patch-clamp technique. They were found to have Ca²⁺-dependent K⁺ channels with the typical properties of the BK channel, i.e. a reversal potential at the calculated equilibrium potential for K⁺ ions, a striking voltage dependence, and a conductance of approximately 200 pS ([K⁺]₀ 50 mM, [K⁺]_i 150 mM, positive patch potentials). Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ currents in heart muscle, reduced the open probability of the BK channels concentration-dependently (1–100 M) when applied at the cytosolic side of membrane inside-out patches. At 100 M [Ca²⁺]_i, the IC₅₀ of tedisamil was 13.8 M (¯x, n=5). Tedisamil increased the frequency of channel closures, and reduced the mean duration of openings from 8 ms to < 1 ms, while the mean duration of closures within bursts (1–2 ms) was not altered. Tedisamil did not affect long closures (> 160 ms) between bursts, either. The mean time of residence of tedisamil at the BK channel was estimated to be 1–2ms. Hence, tedisamil, in comparison to the slow blocker Ba²⁺ and the fast blocker tetraethylammonium, holds the position of an intermediate K⁺ channel blocker.Supported by the Deutsche Forschungsgemeinschaft     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Measurement of intracellular Ca2+ concentration using Indo-1 during simultaneous flash photolysis to release Ca2+ from DM-nitrophen

We have constructed a modular instrument to measure intracellular [Ca²⁺] ([Ca²⁺]_i) in single isolated cells while simultaneously imposing step changes in [Ca²⁺]_i using caged Ca²⁺. By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd: YAG) laser, the instrument can operate with low maintained illumination to measure [Ca²⁺]_i using a ratiometric Ca²⁺-sensitive fluorophore and also activate the release of Ca²⁺ from a caged-Ca²⁺ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca²⁺ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca²⁺]_i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.     

The release of intracellular Ca2+ in lacrimal acinar cells by α-, β-adrenergic and muscarinic cholinergic stimulation: the roles of inositol trisphosphate and cyclic ADP-ribose

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the -adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1–4 phosphate groups, resulting in release of Ca²⁺ from intracellular stores. Stimulation with the -adrenergic agonist phenylephrine, however, causes a release of Ca²⁺ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca²⁺ rise was completely inhibited by 100 M ryanodine. Adrenaline (causing activation of both - and -adrenergic receptors) induces a Ca²⁺ release with inositol phosphate synthesis identical to that occuring in the -adrenergic response. Thus, the signalling pathway for -adrenergic stimulation occurs via a path different from that which releases Ca²⁺ via muscarinic cholinergic and -adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] cause release of Ca²⁺ from intracellular stores. The Ca²⁺ release evoked by cADP-ribose, but not by Ins(1,4,5)P ₃, was abolished by 100 M ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca²⁺ signalling. When the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca²⁺]_i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (Ap _n A) are known to influence cellular Ca²⁺ activity ([Ca²⁺]_i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca²⁺ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca²⁺]_i was measured as a fluorescence ratio F ₃₄₀/F ₃₈₀ with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca²⁺]_i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap₃A–Ap₆A) increased [Ca²⁺]_i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca²⁺]_i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap₃A, Ap₄A, Ap₅A, Ap₆A (each 5 mol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 mol/l, 2.0 mol/l and 4.0 mol/l with Ang II, Ap₃A, ATP and Ap₄A, respectively. Ap₄A (100 mol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca²⁺]_i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.     

α1-Adrenergic regulation of Cl− and Ca2+ movements in rat parotid acinar cells

In rat parotid acinar cells, maximal ₁-adrenergic receptor stimulation (10^–5 M epinephrine +10^–5 M propranolol) leads to a rapid (<10 s), 4–5-fold elevation in cytosolic Ca²⁺ ( 800 nM at peak) which decreases to 50% of peak Ca²⁺ by 3–4 min. Similarly, cells preloaded with³⁶Cl^– show a rapid (<10 s) 35–50% loss of³⁶Cl^– which returns to 80% of resting values in 3–4 min. Both responses are dependent on epinephrine, with half-maximal effects achieved at 2×10^–7 M and 2×10^–6 M agonist for Cl^– and Ca²⁺, respectively. In the presence of low extracellular Ca²⁺ (i.e. with EGTA), the initial rapid changes in cellular Ca²⁺ and Cl^– are unaltered. However, cellular Ca²⁺ and Cl^– levels return to basal values sooner than when extracellular Ca²⁺ is present (within 2 and 3 min, respectively). Maximal epinephrine-induced Ca²⁺ and Cl^– responses are unaffected by the ₂-adrenergic antagonist, yohimbine, are completely blocked by the ₁-adrenergic antagonist, SZL-49, and are similar to ion fluxes induced by maximal muscarinic-cholinergic receptor stimulation (10^–5 M carbachol). The data suggest that a close association exists between mobilization of intracellular Ca²⁺ and Cl^– content in rat parotid acinar cells after ₁-adrenocetor stimulation.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Influence of pH on isometric force development and relaxation in skinned vascular smooth muscle

The effects of pH (from pH values 6.50–7.10) on isometric tension development and relaxation were investigated in Triton X-100 skinned rat caudal artery. Helically cut skinned strips contracted in 21 M Ca²⁺ were studied with respect to maximal isometric tension (P_o) and rate of contraction (T_0.5C), and following relaxation in 18 nm Ca²⁺, the rate of relaxation (T_0.5R). Acidic pH (pH 6.50) decreased P_o to 87% of isometric force obtained at pH 6.90, and increased the rate of contraction as shown by a decrease of T_0.5C to 80%. In contrast, T_0.5R increased 4.5-fold, indicating that with a change of only 0.40 pH units, relaxation rates were dramatically decreased. pCa-tension curves at pH values 6.50, 6.70, 6.90 and 7.10 indicated no significant shift in half maximal activation (pCa₅₀) between pH 6.50 and 6.70, but a significant (P<0.01) shift in pCa₅₀ between pH 6.70 ([Ca²⁺]=0.46 M) and pH 7.10 ([Ca²⁺]=0.87 M). Compared to contractions at pH 6.90, myosin light chain (LC₂₀) phosphorylation at pH 6.50 was significantly greater at 30 and 60 s into contraction but not significantly different at 3–10 min. At both pH 6.50 and 6.90, dephosphorylation was rapid and substantially preceded relaxation; LC₂₀ dephosphorylation and relaxation occurred more rapidly at pH 6.90 than at 6.50. At pH 6.50 and 6.90, relax solutions made with increased Ca²⁺ buffering capacity showed no effect in enhancing T_0.5R, suggesting the difference between relaxation rates was not due to Ca²⁺ diffusion limitations from the skinned strip. We suggest pH changes can after the contractile and relaxation responses in vascular smooth muscle and these effects may be related to LC₂₀ phosphorylation/dephosphorylation regulatory mechanisms.Abbreviations PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - EGTA 5-ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - DTT dithiothreitol - HDTA 1,6-diaminohexane-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate J. P. Gardner was supported in part by an AHA N.J. affiliate postdoctoral fellowship     

Effects of β-adrenoceptor stimulation on intracellular Ca transients and tension in rat ventricular muscle

Effects of -adrenoceptor stimulation on intracellular Ca²⁺ transients and tension were explored in rat ventricular muscles injected with aequorin. Adrenaline (0.05–5.0 M) and isoproterenol (0.05–1.0 M) increased the peak of twitch tension and accelerated relaxation. The former effect depended on Ca²⁺ concentration in Tyrode's solution ([Ca²⁺]_o) and the stage of the experiment. Low concentrations of these drugs added to normal Tyrode's solution containing 2 mM [Ca²⁺]_o did not potentiate twitch tension in the early stage of the experiments. These drugs increased the peak of the aequorin light signal and slightly accelerated the falling phase of the light especially the tail. Effects of dibutyryl-cyclic AMP (DB-cAMP) (0.1–5.0 mM) and 3-isobutyl-1-methylxanthine (IBMX) (0.01–0.5 mM) were qualitatively similar to those of adrenaline and isoproterenol.Isoproterenol applied at the peak of Na-deficient contracture decreased tension without significantly changing the light signal; similar results were obtained in the presence of ryanodine (1 M).The results were interpreted as follows: The increase of intracellular cAMP induced by -adrenoceptor stimulation facilitated Ca²⁺ uptake by sarcoplasmic reticulum (SR) and decreased Ca²⁺ sensitivity of contractile elements. Faster relaxation induced by cAMP was considered to be due to the decrease of Ca²⁺ sensitivity of contractile elements and faster Ca²⁺ uptake by SR. The slightly faster falling phase of light transient might be due to the faster Ca²⁺ uptake by SR, which predominates over the slower fall of [Ca²⁺]_i induced by the decreased Ca²⁺ sensitivity of the contractile element.     

Ca2+ entry through the apical membrane reduces antidiuretic hormone-induced hydroosmotic response in toad urinary bladder

The role of Ca²⁺ in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca²⁺ entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow _H2O) and short circuit current (I _sc) were measured. In most experiments the bladders were treated with small amounts of Ag⁺ (10^–7 mol/l) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkali-earth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca²⁺ is the major cation in the apical solution of these Ag⁺-treated bladders, I _sc is mostly due to Ca²⁺ entry through the apical membrane. Ag⁺ increased I _sc and simultaneously inhibited _H2O in bladders perfused with Ca²⁺ solutions on the apical side. Addition of La³⁺ to the apical solution reversed the stimulation of I _sc and the inhibition of _H2O produced by Ag⁺. When bladders were perfused with Ca²⁺-free solutions on the apical side, addition of Ag⁺ did not inhibit _H2O while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca²⁺ solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (ClPheS-cAMP) the addition of Ag⁺ did not inhibit _H2O while it still increased I _sc. Finally, addition of Ca²⁺ to the apical solution of bladders not treated with Ag⁺ reduced _H2O. These results taken together with other findings in the literature suggest: (1) Ca²⁺ entry through the Ag⁺-treated amiloride-insensitive cation channel of the apical membrane inhibits _H2O; (2) the effects of Ca²⁺ entry are at a regulatory site that precedes the interaction of cAMP with the water channels; (3) it is also possible that Ca²⁺ entry through the unmodified amiloride-insensitive cation channel may have some inhibitory effect on _H2O.     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 M), a blocker of voltage-sensitive Ca²⁺ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2, 7 bis(2-carboxyethyl)-5, 6-carboxyfluorescein (BCECF) and Ca²⁺ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl^–, an acidification of the cytoplasm and an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The latter is inhibited by the Ca²⁺ channel blocker D-600 (1–3 M). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca²⁺ channels which increases [Ca²⁺]_i, thus stimulating the contraction of vascular smooth muscle cells.     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.