Ca2+ ATPase activity in essential and renal hypertension

In 15 patients with essential hypertension, 16 patients with renal hypertension and in 12 healthy subjects Ca2+ ATPase activity was determined in red blood cells both in the basal state and after maximal stimulation with calmodulin. Normal subjects showed a basal and maximal activity of 7.1 +/- 3.6 and 16.0 +/- 2.3 pmol phosphate/min.10(6) RBC, respectively. Renal hypertensives had a similar basal Ca2+ ATPase activity (5.4 +/- 4.1 pmol phosphate/min.10(6) RBC) and a lowered maximal Ca2+ ATPase activity (9.8 +/- 5.4 pmol phosphate/min.10(6) RBC, p < 0.05). In essential hypertensives basal and maximal Ca2+ ATPase activity was 9.0 +/- 5.3 and 35.4 +/- 14.4 pmol phosphate/min.10(6) RBC, respectively, the latter being significantly increased (p < 0.01). This finding, which is in contrast to earlier results indicating a lowered Ca2+ ATPase activity in essential hypertension, may be explained as a consequence of an increased Ca2+ influx in essential hypertension. A lowered Ca2+ ATPase activity does not seem to be involved in the pathogenesis of essential hypertension.     

Polymorphonuclear leukocyte membrane fluidity and cytosolic Ca2+ concentration in diabetes mellitus

OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.     

Sarcoplasmic reticular Ca2+ pump ATPase activity in congestive heart failure due to myocardial infarction

OBJECTIVE: Earlier studies have shown a depression in the sarcoplasmic reticular (SR) Ca2+ uptake and gene expression in Ca2+ pump ATPase protein in congestive heart failure subsequent to myocardial infarction. It is the objective of this study to understand further the mechanisms of depressed SR Ca2+ pump activity in the failing heart. METHODS: Heart failure in rats was induced by occluding the left coronary artery for 16 weeks and the viable left ventricle was processed for the isolation of SR membranes. Sham-operated animals were used as control. The characteristics of SR Ca2+ pump ATPase in the presence of different concentrations of K+, Ca2+ and ATP were examined and the purity of these membranes was monitored by determining the marker enzyme activities. In addition to measuring changes in cyclic adenosine monophosphate (cAMP) protein kinase and Ca(2+)-calmodulin induced phosphorylation, alterations in SR phospholipid composition as well as sulfhydryl (SH) group content were investigated. RESULTS: Ca(2+)-stimulated ATPase activity, unlike Mg(2+)-ATPase activity, was depressed in the left ventricular SR from failing hearts as compared to control. The decrease in Ca(2+)-stimulated ATPase activity was seen at different concentrations of Ca2+, K+ and ATP but no changes in the affinities of the enzyme for Ca2+ and ATP were evident. The SR Ca(2+)-stimulated ATPase activities in the presence of both cAMP-dependent protein kinase and Ca(2+)-calmodulin were markedly decreased in the failing hearts when compared to control preparations. Furthermore, the 32P incorporation in the presence of cAMP-dependent protein kinase or Ca(2+)-calmodulin was also reduced in the experimental heart SR membranes. The phospholipid composition of the SR membranes from the failing heart was markedly altered. No changes in SH-group or the degree of cross contamination with other membranes were apparent in the failing heart SR. CONCLUSIONS: These results suggest that abnormalities in membrane phospholipid composition and phosphorylation of the enzyme may partly explain the observed depression in SR Ca2+ pump ATPase activity in heart failure following myocardial infarction.     

Renal handling of Ca2+ in diabetes

Transient activation of COS-1 cell phospholipase-D (PLD) in response to the protein kinase C (PKC) agonist tetradecanoyl phorbol acetate (TPA) was demonstrated by monitoring the ethanol-dependent accumulation of phosphatidylethanol (PtdEth). Transfection of COS-1 cells with PKC-alpha (wild type and constitutively activated mutants) produced no detectable ptdEth on incubation of transfected cells in the presence of ethanol. However, the response of transfected cells to subsequent TPA stimulation was inhibited, consistent with a role for the PKC-alpha in the suppression of PLD activity.     

Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 microM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 microM PCP in vitro. A significant reduction of SR Ca2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca2+ activation. These results indicate that PCP alters the myocardial Ca2+ homeostasis by inhibiting the Ca2+ ATPase in cardiac SR in rats. Inhibition of SR Ca2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.     

Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes

Proliferation in mammalian cells is controlled primarily in the G1-phase of the cell cycle through the action of the G1 cyclin-dependent kinases, CDK4 and CDK2. To explore the mechanism of cellular response to extrinsic factors, specific loss of function mutations were generated in two negative regulators of G1 progression, p21 and pRB. Individually, these mutations were shown to have significant effects in G1 regulation, and when combined, Rb and p21 mutations caused more profound defects in G1. Moreover, cells deficient for pRB and p21 were uniquely capable of anchorage-independent growth. In contrast, combined absence of pRB and p21 function was not sufficient to overcome contact inhibition of growth nor for tumor formation in nude mice. Finally, animals with the genotype Rb+/-;p21(-/-) succumbed to tumors more rapidly than Rb+/- mice, suggesting that in certain contexts mutations in these two cell cycle regulators can cooperate in tumor development.     

Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase

The Ca2+ transport ATPases of intracellular membranes exhibit an intracellular long-range functional linkage which is the basic mechanistic device for Ca2+ transport through ATP utilization. The functional linkage operates between a phosphorylation (catalytic) domain located in the extramembranous region, and a Ca2+ binding domain located in the membrane bound region of the enzyme. The two domains are separated by a distance of approximately 50 A, and are both affected by binding of a single molecule of the highly specific inhibitor, thapsigargin, to the enzyme. Functional and structural features are here described to explain the long-range linkage through the protein structure.     

Ca2+ translocation across sarcoplasmic reticulum ATPase randomizes the two transported ions

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5 degrees C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation.     

PLA2 activity regulates Ca2+ storage-dependent cellular proliferation

The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca2+). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca2+ was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca2+ measurements were performed in the absence of extracellular Ca2+ to identify the contribution of intracellular Ca2+ sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca2+] while depleting internal Ca2+ stores. These Ca2+ changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca2+ stores. Ca2+ is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca2+-dependent mechanisms.     

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.     

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement

The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.     

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.     

Behavior of the polymorphonuclear leukocyte membrane fluidity and cystolic Ca2+ content in vascular atherosclerotic disease with and without non-insulin-dependent diabetes mellitus

In 71 subjects with vascular atherosclerotic disease (VAD), in 32 VAD subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in 31 normal controls, we evaluated polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content. The PMN membrane fluidity was obtained by marking intact and unstimulated PMN cells with fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and the PMN cytosolic Ca2+ content was obtained by marking intact and unstimulated PMN cells with the fluorescent probe Fura 2-AM. From the obtained results, it is evident that PMN membrane fluidity does not differentiate normals from VAD subjects and VAD subjects with NIDDM, and normals from subjects with monovascular disease (MVAD) and polyvascular disease (PVAD) with and without NIDDM. The PMN cytosolic Ca2+ content is significantly increased in VAD subjects and VAD subjects with NIDDM, and also in MVAD and PVAD subjects with and without NIDDM. A positive correlation is present between PMN membrane fluidity and PMN cytosolic Ca2+ content in normals and VAD subjects, but not in VAD subjects with NIDDM. In conclusion, in VAD subjects with and without NIDDM, an increase of the PMN cytosolic Ca2+ content is present; this increase might be related to the PMN spontaneous activation.     

Saltatory propagation of Ca2+ waves by Ca2+ sparks

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

Ca2+ induces an increase in cGMP-phosphodiesterase activity in squid retinal photoreceptors

In this paper we describe a rapid, isocratic high performance liquid chromatography (HPLC) method for the study of radioactive fatty acid incorporation into complex lipids of human erythrocytes, which allows the simultaneous separation of the major phospholipid classes and long-chain acylcarnitines. The lipid extract of erythrocytes pulsed with radioactive fatty acids was injected into an HPLC system equipped with a silica column. The individual components eluted were monitored by ultraviolet absorption and radioactive emission. With respect to the UV profile, the radioactive profile showed an additional peak between phosphatidyl-choline and phosphatidylethanolamine, which was identified as long-chain acylcarnitine by different experimental approaches. The radioactivity recovered in the long-chain acylcarnitines contains essential information enabling definition of acyl trafficking in red cells.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels

The three subtypes of inositol trisphosphate (InsP3) receptor expressed in mammalian cells are each capable of forming intracellular Ca2+ channels that are regulated by both InsP3 and cytosolic Ca2+. The InsP3 receptors of many, though perhaps not all, tissues are biphasically regulated by cytosolic Ca2+: a rapid stimulation of the receptors by modest increases in Ca2+ concentration is followed by a slower inhibition at higher Ca2+ concentrations. Despite the widespread occurrence of this form of regulation and the belief that it is an important element of the mechanisms responsible for the complex Ca2+ signals evoked by physiological stimuli, the underlying mechanisms are not understood. Both accessory proteins and Ca2+-binding sites on InsP3 receptors themselves have been proposed to mediate the effects of cytosolic Ca2+ on InsP3 receptor function, but the evidence is equivocal. The effects of cytosolic Ca2+ on InsP3 binding and channel opening, and the possible means whereby the effects are mediated are discussed in this review.