Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca(2+)](i) rise were investigated by high-speed Ca(2+) imaging during early development of monospermic eggs. Every [Ca(2+)](i) rise involved a Ca(2+) wave. In the first Ca(2+) transient, [Ca(2+)](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca(2+) release mechanisms. The first step was a Ca(2+) wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, approximately 20 microm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca(2+)](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca(2+) wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca(2+) oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation ( approximately 3 h after fertilization). (1) The duration of Ca(2+) transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca(2+)](i) rise became greater and wave speeds increased up to 80-100 microm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca(2+) wave, although its displacement became smaller. (5) The Ca(2+) wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.     

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

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

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

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

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

beta-Adrenergic activation reveals impaired cardiac calcium handling at early stage of diabetes

Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that beta-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the beta-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca(2+)](i) levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca(2+)](i) transient the kinetic parameters of the sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca(2+)](i) transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca(2+)](i) amplitude and rate of rise and decline of [Ca(2+)](i) as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under beta-adrenoceptor stimulation. Upon beta-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca(2+)](i) amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+)-release channel in response to beta-adrenoceptor challenge. Alterations in Ca(2+)(i) handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.     

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.     

Activation and propagation of Ca(2+) release during excitation-contraction coupling in atrial myocytes

Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.     

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.     

Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.     

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.     

Physiological and pathological modulation of ryanodine receptor function in cardiac muscle

Calcium release from the sarcoplasmic reticulum (SR) in cardiac muscle occurs through a specialised release channel, the ryanodine receptor, RyR, via the process of Ca-induced Ca release (CICR). The open probability of the RyR is increased by elevation of cytoplasmic Ca concentration ([Ca(2+)](i)). However, in addition to Ca, other modulators affect the RyR open probability. Agents which increase the RyR opening during systole produce a transient increase of systolic [Ca(2+)](i) followed by a return to the initial level due to a compensating decrease of SR Ca content. Increasing RyR opening during diastole decreases SR Ca content and thereby decreases systolic [Ca(2+)](i). We therefore conclude that potentiation of RyR opening will, if anything, decrease systolic [Ca(2+)](i). The effects of specific examples of modulators of the RyR, such as phosphorylation, metabolic changes, heart failure and polyunsaturated fatty acids, are discussed.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.     

Essential role for calcium waves in migration of human vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca(2+) orchestrate the migratory cycle in human vascular SMCs. We carried out Ca(2+) imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca(2+) waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca(2+) waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca(2+)](i). To investigate a role for Ca(2+) waves in the migratory cycle, we loaded cells with the Ca(2+) chelator BAPTA, which abolished Ca(2+) waves and significantly reduced retraction, supporting a causal role for Ca(2+) in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca(2+) oscillations was reduced when Ca(2+) release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca(2+) stores in generation of waves. We conclude that Ca(2+) waves are essential for migration of human vascular SMCs and can encode cell polarity.     

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.