Ca(2+)-induced Ca(2+) release in the pancreatic beta-cell: direct evidence of endoplasmic reticulum Ca(2+) release

The role of the Ca(2+)-induced Ca(2+) release channel (ryanodine receptor) in MIN6 pancreatic beta-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca(2+). Depolarization of MIN6 cells with KCl led to release of Ca(2+) from the ER. This ER Ca(2+) release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca(2+) channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca(2+) was also inhibited by ryanodine, which did not alter voltage-gated Ca(2+) channel activation. Both ER and cytoplasmic Ca(2+) changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca(2+) but no detectable change in ER Ca(2+). Carbamyl choline caused ER Ca(2+) release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca(2+)-induced Ca(2+) release augments cytoplasmic Ca(2+) signals in pancreatic beta-cells.     

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.     

Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis. Protein S Study Group

Protein S is a protein C-dependent and independent inhibitor of the coagulation cascade. Deficiency of protein S is an established risk factor for venous thromboembolism. We have used a strategy of specific amplification of the coding regions and intron/exon boundaries of the active protein S gene (PROS1) and direct single-strand solid phase sequencing, to seek mutations in 35 individuals with phenotypic protein S deficiency. Nineteen point mutations (16 novel) in 19 probands (or relatives of probands) with venous thromboembolism are reported here. Fifteen of the 19 mutations were expected to be causal and included 10 missense mutations (Lys9Glu, Glu26Ala, Gly54Glu, Cys145Tyr, Cys200Ser, Ser283Pro, Gly340Asp, Cys408Ser, Ser460Pro, and Cys625Arg). Three of the 15 mutations resulted in premature stop codons (delete T 635 producing a stop codon at position 126, Lys368stop and Tyr595stop) and two were at intron/exon boundaries (+1 G to A in intron d and +3 A to C in intron j). Of the remaining four mutations, three were within intronic sequence and one was a silent mutation within the coding region and did not alter amino acid composition. In two of the 10 missense mutations, reduced plasma protein S activity compared with antigen level suggested the presence of variant (type II) protein S.     

Privileged coupling between Ca(2+) entry through plasma membrane store-operated Ca(2+) channels and the endoplasmic reticulum Ca(2+) pump

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is the third element of capacitative calcium entry. It colocalizes with STIM1 and Orai1 at puncta, where couples plasma membrane store-operated Ca(2+) channels (SOC) to Ca(2+) pumping into the ER. The efficiency of this calcium entry-calcium refilling (CECR) coupling is comparable to the classic excitation-response transduction mechanisms. This allows efficient filling of the endoplasmic reticulum (ER) with the Ca(2+) entering through SOC channels with little progression of the Ca(2+) wave towards the cell core. CECR coupling is very sensitive to changes in stoichiometry among STIM, Orai and SERCA, with excess Orai antagonizing ER refilling. ER takes up most of the calcium load that enters through SOC, whereas mitochondria take up a very small fraction. This difference is due to the spatial positioning with regard to SOC, the amplitude of the high Ca(2+) microdomains, and the differences in the Ca(2+) affinity of the uptake mechanisms.     

Metabotropic Ca(2+) channel-induced Ca(2+) release and ATP-dependent facilitation of arterial myocyte contraction

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.     

Growth-dependent alterations of intracellular Ca(2+)-handling mechanisms of vascular smooth muscle cells. PDGF negatively regulates functional expression of voltage-dependent, IP3-mediated, and CA(2+)-induced Ca2+ release channels.

To examine the alterations of intracellular Ca2+ ([Ca2+]i)-handling mechanisms in cultured vascular smooth muscle cells (VSMCs) of rat aorta (Shin et al Circ Res 1991;69:551-556), we stimulated VSMCs by extracellular high K+, caffeine, and angiotensin II and evaluated Ca2+ influx through voltage-dependent Ca2+ channels, Ca(2+)-induced Ca2+ release, and inositol trisphosphate-dependent Ca2+ release from internal stores. Percentage of VSMCs responding to each stimulant (responder ratio) and degree of [Ca2+]i increase in the responding cells were analyzed by a two-dimensional fura-2 imaging system. The responder ratios to the three stimulants were high (70-90%) in the quiescent phase (days 1-2), although some cells selectively responded to one or two of the stimulants. Responder ratios prominently decreased to approximately 20% in the proliferating phase (days 2.5-3). In the subconfluent (days 3.5-4) and postconfluent (days 5-6) phases, the responder ratio to high K+ and angiotensin II recovered to the same level as during the quiescent phase, whereas that to caffeine remained low (approximately 10-20%). In responding cells, the degree of [Ca2+]i increase by caffeine and angiotensin II was stable (approximately 100%) during culturing, whereas that to high K+ was small (approximately 30-40%) in the quiescent and proliferating phases and rapidly increased threefold in the subconfluent and postconfluent phases. Furthermore, arrest of cell growth in serum-free medium prevented the reduction of responder ratios in the proliferating phase and restored the decreased ratio of the caffeine responder. Acceleration of VSMC proliferation by platelet-derived growth factor decreased the ratios in all phases. These results imply that 1) the functional expressions of [Ca2+]i-handling mechanisms in response to these vasoactive stimuli are influenced by cell growth and cytodifferentiation of VSMCs or platelet-derived growth factor and 2) they are regulated independently from each other.     

Ca(2+) transients and Ca(2+) waves in purkinje cells : role in action potential initiation

Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces a biphasic Ca(2+) transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca(2+) transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM, and studied in normal Tyrode's solution (24 degrees C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca(2+)](i) were obtained from fluorescent images with an epifluorescent microscope (x20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L(0)) compared with resting levels. This was followed by a further increase in fluorescence (L(1)) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 microm/s). In 62% of the aggregates, initial fluorescent changes of L(0) were followed by focally arising Ca(2+) waves (L(2)), which propagated at 158+/-14 microm/s (n=13). Spontaneous Ca(2+) waves (L(2)*) propagated like L(2) (164+/-10 microm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L(2)* elicited action potentials. Both spontaneous Ca(2+) wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca(2+) waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca(2+) through I(CaL) and release from a subsarcolemmal compartment (L(0)). Ca(2+) release during L(0) either induced further Ca(2+) release, which propagated toward the cell core (L(1)), or initiated Ca(2+) release from small regions and caused L(2) Ca(2+) waves, which propagated throughout the aggregate. Spontaneous Ca(2+) waves (L(2)*) induce action potentials.     

Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-content in isolated rat and rabbit myocardium

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca(2+)-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 degrees C. In addition, the frequency-dependent relative contribution of SR Ca(2+)-uptake and Na(+)/Ca(2+)-exchange for cytosolic Ca(2+)-removal was assessed by paired RCCs. With increasing rest intervals (1-240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45+/-10% and 61+/-11%, respectively P<0. 05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67+/-16% at 240 s P<0.05) and RCC amplitude (by 20+/-14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295+/-50% (P<0.05) and RCC amplitude by 305+/-80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43+/-7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16+/-7%). The SR Ca(2+)-uptake relative to Na(+)/Ca(2+)-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (;66+/-2% at all frequencies). We conclude that increased SR Ca(2+)-load contributes to the positive force-frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca(2+)-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force-frequency relationship in rats. SR Ca(2+)-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.     

Interactions between Ca(2+) and H(+) and functional consequences in vascular smooth muscle

Ca(2+) and H(+) ions can profoundly alter vascular tone. In many physiological and pathological processes, changes in the concentration of both ions occur. Thus, to understand the processes and mechanisms that modify force, it is necessary to understand what changes occur in these ions and, importantly, how they interact with each other. In this minireview, we highlight the quantitatively important mechanisms involved in the contractile responses of vascular tissues to pH change and discuss the cellular and molecular reasons underlying these responses.     

Passive transport pathways for Ca(2+) and Co(2+) in human red blood cells. (57)Co(2+) as a tracer for Ca(2+) influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1μM, the progress curve for (45)Ca uptake at 1mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca(T)](c) to reach at [Ca(T)](c) about 5μmol(lcells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca(T)](c). Moreover, vanadate markedly increases the passive Ca(2+) influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40-50μmol(lcells)(-1)h(-1). The apparent Ca(2+) affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the (45)Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1mM Ca(2+) estimated at 50-60μmol(lcells)(-1)h(-1). The Ca influx increases with the extracellular Ca(2+) concentration with a saturating component, with K(?(Ca)) about 0.3mM, plus a non-saturating component. From (45)Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The (45)Ca efflux is markedly accelerated by external Ca(2+), both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The (57)Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co(2+) is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The (57)Co uptake shows several similarities with the (45)Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J(max) about 16μmol(lcells)(-1)h(-1) and K(?(Co)) about 0.1mM, plus a non-saturating component. The (57)Co and (45)Ca uptake shows mutual inhibition, and at least the stochastic Ca(2+) influx is inhibited by Co(2+). The (57)Co and (45)Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100μM. The (57)Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The (57)Co influx amounts to about half the (45)Ca influx in ATP-depleted cells. It is speculated that the basal Ca(2+) and Co(2+) uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that (57)Co could be useful as a tracer for at least the channel-like Ca(2+) entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca(2+) buffering. The molecular identity of the putative common transporter(s) remains to be defined.     

Reduced sarcoplasmic reticulum Ca(2+) load mediates impaired contractile reserve in right ventricular pressure overload

Myocardial contractile reserve is significantly attenuated in patients with advanced heart failure. The aim of this study was to identify mechanisms of impaired contractile reserve in a large animal model that closely mimics human myocardial failure. Progressive right ventricular hypertrophy and failure were induced by banding the pulmonary artery in kittens. Isometric contractile force was measured in right ventricular trabeculae (n=115) from age-matched Control and Banded feline hearts. Rapid cooling contractures (RCC) were used to determine sarcoplasmic reticulum (SR) Ca(2+) load while assessing the ability of changes in rate, adrenergic stimulation and bath Ca(2+) to augment contractility. The positive force-frequency relationship and robust pre- and post-receptor adrenergic responses observed in Control trabeculae were closely paralleled by increases in RCC amplitude and the RCC2/RCC1 ratio. Conversely, the severely blunted force-frequency and adrenergic responses in Banded trabeculae were paralleled by an unchanged RCC amplitude and RCC2/RCC1 ratio. Likewise, supraphysiologic levels of bath Ca(2+) were associated with severely reduced contractility and RCC amplitude in Banded trabeculae compared to Controls. There were no differences in myofilament Ca(2+) sensitivity or length-dependent increases in contractility between Control and Banded trabeculae. There was a significant decrease in SR Ca(2+)-ATPase pump abundance and phosphorylation of phospholamban and ryanodine receptor in Banded trabeculae compared with Controls. A reduced ability to increase SR Ca(2+) load is the primary mechanism of reduced contractile reserve in failing feline myocardium. The similarity of impaired contractile reserve phenomenology in this feline model and transplanted hearts suggests mechanistic relevance to human myocardial failure.     

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.     

Vanadate-induced Ca(2+) and Co(2+) uptake in human red blood cells

The vanadate-induced increase in passive uptake of calcium and cobalt and their interference were studied in human red cells using ⁴⁵Ca and ⁵⁷Co as tracers. Vanadate is a potent inhibitor of the Ca-pump in red cells, although in fed cells a residual pump activity remains that is highly significant compared to the passive influx, and even in cells that are both ATP-depleted and vanadate-treated the pump arrest is not complete. In the presence of vanadate the Ca^2 + uptake is increased due to inhibition of Ca-pump extrusion, but is further increased due to a vanadate-induced increment in passive influx. In order to measure the vanadate-induced increment in Ca^2 + influx, the total uptake in vanadate-treated cells is corrected for the basal influx, as recorded in ATP-depleted cells in the presence of tetrathionate (5 mM) that has been shown to eliminate the residual Ca-pump activity in ATP-depleted cells. The ⁵⁷Co uptake is also increased by vanadate. ⁵⁷Co is not transported by the Ca-pump, and hence the uptake in vanadate-treated cells can be directly compared to the basal uptake, both in fed and in ATP-depleted cells. The vanadate effect shows rapid onset and appears to be irreversible. The vanadate-induced increment in uptake of both ⁴⁵Ca and ⁵⁷Co is reduced by about 50% in ATP-depleted cells compared to fed cells, suggesting a metabolism- or SH-group-dependent component. The influx of both ⁴⁵Ca (in ATP-depleted cells) and ⁵⁷Co (in fed cells) increases with the vanadate concentration, with a similar K_½ (0.4 and 0.3 mM, respectively), and is nearly maximal at 5 mM vanadate. The vanadate-induced increment in influx of both ⁴⁵Ca and ⁵⁷Co increases with the extracellular concentration as a saturable function, with K_½ estimated at, respectively, 700 and 80 μM. In the case of ⁵⁷Co K_½ is similar in fed and in ATP-depleted cells. The vanadate-induced uptake of ⁴⁵Ca and of ⁵⁷Co shows interference. The uptake of ⁴⁵Ca is inhibited by Co^2 +, and the uptake of ⁵⁷Co is inhibited by Ca^2 +, although with an unexplained time course. The vanadate-induced uptake of ⁴⁵Ca and ⁵⁷Co are both inhibited, and to a similar degree, by the 1,4-dihydropyridine Ca^2 +-channel blocker nifedipine, although only at concentrations much higher than IC₅₀ for classical Ca-channels. The vanadate-induced increment in ⁵⁷Co uptake is electroneutral, in contrast to the basal uptake that is at least partially electrogenic. In experiments with resealed ghosts a vanadate-induced ⁵⁷Co uptake could not be detected. The vanadate-induced increment in ⁵⁷Co uptake amounts to nearly half the increment in ⁴⁵Ca uptake, both in fed and in ATP-depleted cells. It is speculated that the vanadate-induced Ca^2 + and Co^2 + uptake could be mediated by a putative common transporter, which appears to be separate and distinct from the putative common transporter for basal Ca^2 + and Co^2 + uptake.     

Effects of cytosolic ATP on Ca(2+) sparks and SR Ca(2+) content in permeabilized cardiac myocytes

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca(2+) sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca(2+) sparks. In the presence of 20 micromol/L ATP, the amplitude increased by 48.7+/-10.9%, and the frequency decreased by 77.07+/-3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca(2+) sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca(2+) content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca(2+) sparks. This was accompanied by a rapid decrease in SR Ca(2+) content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca(2+) content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca(2+) sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca(2+) channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca(2+) overload occur.     

Local Ca(2+) signaling and EC coupling in heart: Ca(2+) sparks and the regulation of the [Ca(2+)](i) transient

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.