The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle

Treatment of rat ventricular cells with 10 mM EGTA makes the sarcolemma highly permeable to small ions and molecules without removing its restriction of the diffusion of larger molecules or inactivating all of its enzymatic functions. These hyperpermeable cardiac cells have been used to study the regulation of the range of concentration of Ca over which activation of the contractile proteins occurs (Ca sensitivity). The Ca sensitivity can varied from three- to sixfold without any significant alteration in the general shape of the relation between force and Ca concentrations. Although cyclic nucleotides in concentrations of 10(-9) to 10(-5) M do not influence Ca sensitivity, in the presence of a phosphodiesterase inhibitor, cGMP increases and cAMP decreases Ca sensitivity. Treatment of the hyperpermeable cells with a nonionic detergent raises Ca sensitivity as does removal of the phosphate donor by complete substitution of CTP for ATP. These data indicate that Ca sensitivity is probably modulated by a cAMP-dependent phosphorylation that decreases Ca sensitivity. The sarcolemma is required for this reaction to take place. The effect of this reaction is antagonized by a cGMP-dependent reaction occurring inside the cell. Studies involving the perfusion of the heart with and without epinephrine before the exposure to EGTA indicate that epinephrine can regulate this system of control of Ca sensitivity. The functional considerations of this regulatory system are discussed.     

Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle

The contractile system of rat cardiac muscle that has been made hyperpermeable by soaking the tissue in EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764) can be probed directly with Ca buffer from the bathing solution without significant interference from either sarcoplasmic reticulum or mitochondria on the Ca concentration. Changes in Ca-activated force are due therefore to changes in the properties of the contractile system itself and not to regulation of Ca concentration. The addition of cAMP, cGMP, and GTP, guanylyl imidodiphosphate (GMP-PNP), or epinephrine to the bath does not alter maximum Ca-activated force, but when these drugs are added with 1% nonionic detergent to the bath, contractility increases by as much as 180%. An inhibitor of phosphodiesterase must be present for the inotropic effect of cAMP but not cGMP, GTP, GMP-PNP, or epinephrine. The inotropic response to cAMP is independent of the Ca sensitivity of the contractile system, but guanine nucleotides enhance contractility only when Ca sensitivity is not high. The inotropic effect of epinephrine is inhibited to a large extent by cGMP but not by GMP-PNP. These data can be explained by a model in which contractility is enhanced by a cAMP-regulated phosphorylation that can be controlled through the beta-receptor adenylate cyclase complex in the sarcolemma. The regulation involves two reactions, one a phosphorylation and a second that occurs in the presence of detergent. Phosphorylation of neither the myosin light chain nor the inhibitory subunit of troponin appears to be involved in this mechanism for regulating contractility.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Cell cycle control by Ca2+ in Saccharomyces cerevisiae

We established an experimental system suitable for study of cell cycle regulation by Ca2+ in the yeast Saccharomyces cerevisiae. Systematic cell cycle analysis using media containing various concentrations of Ca2+, a Ca2(+)-ionophore (A23187), and a Ca2(+)-chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) revealed that simultaneous addition of 10 microM A23187 and 10 mM EGTA to cells growing in a Ca2(+)-deficient medium at 22 degrees C caused rapid decrease in intracellular Ca content and resulted in transient G1 arrest followed by block mostly at G2/M, as revealed by flow cytometry. Recovery from G1 arrest was not due to coordinated initiation of DNA synthesis and bud emergence: unbudded cells with S or G2/M DNA were observed. Examination of terminal phenotype suggested that Ca2+ was required at all the stages of the cell cycle except for the initiation of DNA synthesis. The intracellular cAMP level decreased within 10 min of addition of A23187 and EGTA. No significant transient G1 arrest was observed in cells incubated with 8-Br-cAMP, or RAS2val19 and delta bcy1 mutants, which produce a high level of cAMP and have constitutively activated cAMP-dependent protein kinase, respectively. These results indicate that Ca2+ is essential for cell cycle progression and suggest that Ca2+ may regulate the cAMP level. This system will be useful for genetic and molecular studies on cell cycle events regulated by Ca2+.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells

Bundles of cells from rat right ventricular myocardium were made "hyperpermeable" by an overnight soak in 10 mM EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764). In this preparation the cytoplasmic concentration of Ca++ and ATP could be controlled while sarcolemmal receptors and enzymes were retained. The Ca sensitivity of the tissues (as indicated by the pCa for 50% maximum activation) was altered to different extents in the presence of [32Pgamma]ATP by treatment with cyclic nucleotides, catecholamines, or a low concentration of nonionic detergent. The proteins of the tissue were then isolated by SDS-polyacrylamide gel electrophoresis, and the identity of 32P-labeled proteins was determined. The Ca sensitivity is inversely related to the relative amount of 32P incorporated into the inhibitory subunit of troponin (TNI). Extrapolation of the relation to the lowest Ca sensitivity observed gives a stoichiometry of about 0.8 mol PO4 per mol TNI. These results support the hypothesis that Ca sensitivity of cardiac myofibrils is regulated by a phosphrylation of TNI that is stimulated by cyclic AMP (cAMP) and inhibited by cGMP.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca²⁺-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca²⁺-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of ⁴⁵Ca from cells preequilibrated with ⁴⁵Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular ⁴⁵Ca level. Inositol trisphosphate, but not cAMP, evoked ⁴⁵Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Amylase secretion from saponin-permeabilized parotid cells evoked by cyclic AMP

Adenosine 3',5'-monophosphate (cAMP) evoked amylase release from saponin-permeabilized parotid cells of the rat. Saponin concentration was optimal at 10 micrograms/ml. Amylase release was stimulated by cAMP almost as well in Ca2+-free medium containing 1 mM EGTA as in the medium containing a physiological concentration of calcium. Although the basal and stimulated releases of amylase were markedly reduced by the further addition of 5 mM EGTA, the effect of cAMP was still detectable. The half-maximal dose of cAMP was 0.3 mM, whereas those of dibutyryl cAMP and 8-bromo-cAMP were 10-fold lower than that of cAMP. In the presence of 10 microM 3-isobutyl-1-methylxanthine, the half-maximal dose of cAMP was also decreased by 5-fold. These results suggest: 1) intracellular calcium is not essential for the exocytosis of amylase stimulated by cAMP; 2) the responsiveness of the cells to exogenous cAMP is reduced by phosphodiesterase.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Modulation of postjunctional cholinergic sensitivity of rat diaphragm muscle by cyclic adenosine monophosphate.

Addition of 2.5 mM cyclic adenosine monophosphate (cAMP) to the solution bathing a rat diaphragm muscle alters the magnitude of depolarization responses to iontophoretic pulses of acetylcholine (ACh) at neuromuscular endplates. Alterations are repeatable with small variability on a given preparation for initial and repeat experiments on both hemidiaphragms, but are different on each preparation. Five min after addition of the nucleotide solution, increases (potentiations) of up to 30% above control levels and decreases (attenuations) to 50% below control levels are observed. The effects on sensitivity to ACh of dibutyryl cAMP (1.25 mM), monobutyryl cAMP (0.25 mM), and cAMP (2.5 mM) in Ca++ -free solution are a function of whether the experiment is an initial one on that preparation or a repeat experiment after 10 or more minutes of perfusion flow. In all three cases, initial exposure attenuates sensitivity (means at 5 min: --30, --10, and --20%, respectively) and repeat exposure potentiates sensitivity (mean: 20% at 5 min, 15% at 5 min, and 10% at 2 min respectively). A concentration of dibutyryl cAMP (0.25 mM) which is without effect on sensitivity alone, produces a large, transient potentiation (mean: 45% at 1 min) in conjunction with 0.5 mM theophylline. A decrease in the rate of desensitization is observed during exposure to 0.25 mM cAMP. The results are interpreted in terms of a physiological mechanism whereby receptor activity at the postjunctional membrane is modulated by cAMP formed from prejunctionally released ATP.     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.     

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.     

Interaction of cyclic AMP and Ca2+ in the control of electrical coupling in heart fibers

The influence of intracellular injection of cAMP on the electrical coupling of canine Purkinje cells was investigated. It was found that the nucleotide enhanced reversibly the cell-to-cell communication through an increase in junctional conductance. Dibutyryl cAMP (5 X 10(-4) M) plus theophylline (0.4 mM) decreased appreciably the intracellular longitudinal resistance (ri). The interactions of cAMP and Ca on the electrical coupling were also investigated. The nucleotide and Ca have opposite effects on the electrical coupling. In the presence of high [Ca2+]o solutions (6 mM), the intracellular injection of cAMP causes a transient increase in the coupling coefficient followed by an appreciable decrease in cell-to-cell coupling. This reduction in intracellular communication was reversed by injecting EGTA into the same cell. The results of this study support the view that cAMP is a modulator of junctional conductance in cardiac muscle and that the compound interacts with Ca in the control of intracellular communication.     

Meiotic resumption in response to luteinizing hormone is independent of a Gi family G protein or calcium in the mouse oocyte

The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.