Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells

Abstract

Context: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca²⁺ signalling in vascular smooth muscle cells (VSMC). Objective: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca²⁺ concentration [Ca²⁺]i. RGS2 involvement in VSMC proliferation had not been examined. Methods: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca²⁺]i before and after UTP and thapsigargin. [³H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca²⁺ transporters was performed in primary aortic VSMC. Results and discussion: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca²⁺ homeostasis, RGS2KO VSMC showed lower resting [Ca²⁺]i but higher thapsigargin-induced [Ca²⁺]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca²⁺ ATPase-4 (PMCA4) and Na⁺ Ca²⁺ Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca²⁺ ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca²⁺ efflux activity, ⁴⁵Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La³⁺, but not by replacing extracellular Na⁺ with choline, implicating differences in the activity of PMCA and not NCX. Conclusion: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca²⁺-handling mechanisms in response to PDGF stimulation.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

TGF-beta-induced protein beta ig-h3 is upregulated by high glucose in vascular smooth muscle cells

TGF-beta-induced gene-h3 (beta ig-h3) is an adhesive molecule that interacts with integrins. Because TGF-beta plays an important role in diabetic complications and beta ig-h3 serves as a cell substrate, we hypothesized that diabetic conditions might increase beta ig-h3 synthesis in vascular smooth muscle cells (VSMCs), which may subsequently contribute to the pathogenesis of diabetic angiopathy. The concentrations of beta ig-h3 and TGF-beta were measured in conditioned media using an enzyme-linked immunosorbent assay. An immunohistochemical study showed that beta ig-h3 was expressed in the VSMCs and the matrix of rat aortas. TGF-beta stimulated beta ig-h3 production, and high glucose induced beta ig-h3 as well as TGF-beta production in the VSMCs. The high glucose-induced beta ig-h3 expression was almost entirely blocked by an anti-TGF-beta antibody. beta ig-h3 protein mediated the adhesion, spreading, migration, and proliferation of rat VSMCs. These results suggest that the high glucose-induced beta ig-h3 in VSMCs regulates VSMC functions and may play an important role in diabetic angiopathy.     

Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) plays a critical role in generation of complex Ca(2+) signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP(3)R channels were consistently detected with regulation by cytoplasmic InsP(3) and free Ca(2+) concentrations ([Ca(2+)](i)) very similar to that observed for vertebrate InsP(3)R. Long channel activity durations of the Sf9-InsP(3)R have now enabled identification of a novel aspect of InsP(3)R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP(3)R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P(o)) within each mode of 0.007 +/- 0.002, 0.24 +/- 0.03, and 0.85 +/- 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca(2+)](i) and [InsP(3)] does not substantially alter channel P(o) within a mode. Instead, [Ca(2+)](i) and [InsP(3)] affect overall channel P(o) primarily by changing the relative probability of the channel being in each mode, especially the high and low P(o) modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP(3)R channel activity, with implications for the kinetics of Ca(2+) release events in cells.     

Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells

Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output.     

Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells

The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.     

Pathophysiology of voltage-gated K channels in vascular smooth muscle cells: Modulation by protein kinases

In this review, the pathological alteration and clinical relevance of voltage-gated K⁺ (Kv) channels and their specific regulation by protein kinase-dependent signaling in vascular smooth muscle cells are described, particularly focusing on the pulmonary vasculature. The physiological relevance, channel characteristics, pharmacological modulation, and expression of Kv channels vary between different arterial beds and between subdivisions of arteries within those vascular beds. Although detailed signaling cascades regulating Kv channels are not clearly elucidated, it is known that the Kv channels in vascular smooth muscle cells can be tightly regulated by protein kinases C (PKC) and A (PKA). Alterations in Kv channel expression and function has been noted in pathological and pathophysiological conditions including hypertension (pulmonary and systemic), in diabetes and in individuals subjected to prolonged hypoxia (high altitude living). Vascular Kv channels are potential therapeutic targets in diseases such as pulmonary arterial hypertension and, therefore, it is important to understand the specific pharmacological modulation of Kv channel isoforms in different vascular beds.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps

The regulation of cytosolic Ca2+ homeostasis is essential for cells, and particularly for vascular smooth muscle cells. In this regulation, there is a participation of different factors and mechanisms situated at different levels in the cell, among them Ca2+ pumps play an important role. Thus, Ca2+ pump, to extrude Ca2+; Na+/Ca2+ exchanger; and different Ca2+ channels for Ca2+ entry are placed in the plasma membrane. In addition, the inner and outer surfaces of the plasmalemma possess the ability to bind Ca2+ that can be released by different agonists. The sarcoplasmic reticulum has an active role in this Ca2+ regulation; its membrane has a Ca2+ pump that facilitates luminal Ca2+ accumulation, thus reducing the cytosolic free Ca2+ concentration. This pump can be inhibited by different agents. Physiologically, its activity is regulated by the protein phospholamban; thus, when it is in its unphosphorylated state such a Ca2+ pump is inhibited. The sarcoplasmic reticulum membrane also possesses receptors for 1,4,5-inositol trisphosphate and ryanodine, which upon activation facilitates Ca2+ release from this store. The sarcoplasmic reticulum and the plasmalemma form the superficial buffer barrier that is considered as an effective barrier for Ca2+ influx. The cytosol possesses different proteins and several inorganic compounds with a Ca2+ buffering capacity. The hypothesis of capacitative Ca2+ entry into smooth muscle across the plasma membrane after intracellular store depletion and its mechanisms of inhibition and activation is also commented.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Modulation of calcium channels in arterial smooth muscle cells by dihydropyridine enantiomers

The actions of the optical enantiomers of BAY K 8644 and Sandoz 202,791 were studied on barium inward currents recorded using the whole-cell configuration of the patch clamp technique from enzymatically isolated smooth muscle cells from the rabbit ear artery. The enantiomers were applied by bath perfusion or rapidly by a concentration jump technique, which enabled the study of drug action under equilibrium and nonequilibrium conditions. A larger effect of agonists was seen on peak inward current in 110 mM Ba when small rather than large depolarizations were applied. The midpoint voltage of the steady-state inactivation curve of IBa was -12.8 +/- 1.9 mV (n = 4) in the absence of drug, -16.4 +/- 2.5 mV (n = 4) in 1 microM (+)202,791, and -31.4 +/- 0.4 mV (n = 4) in 1 microM (-)202,791. The rate of onset of action of the agonist and antagonist enantiomers of BAY K 8644 and Sandoz 202,791 was studied by rapid application during 20-ms depolarizing steps from different holding potentials to +30 mV at 1 or 0.2 Hz. The drugs were applied as concentration jumps between two single pulses of a pulse train. The rates of onset of drug action on peak IBa during a 1-Hz pulse train were concentration dependent over the range of 100 nM-3 microM for both (+) and (-)202,791. The rate of onset of inhibition of peak current by antagonist enantiomers was not significantly influenced by the test pulse frequency. At a holding potential of -60 mV, the onset rate of the increase in peak IBa on application of 1 microM of agonist enantiomers (+)202,791 or (-)BAY K 8644 during a train of pulses occurred with mean time constants of 2.1 +/- 0.7 s (n = 7) and 2.3 +/- 0.2 s (n = 4), respectively. The onset of current increase on application of 1 microM (+)202,791 during a single voltage clamp step to 20 mV was faster, with a mean time constant of 380 +/- 80 ms (n = 3).     

Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5-trisphosphate in cultured vascular smooth muscle cells

Inositol 1,4,5-trisphosphate (IP3) rapidly increased 45Ca2+ efflux from a nonmitochondrial organelle in cultured vascular smooth muscle cells that were permeabilized with saponin. A nucleotide, preferably ATP, was essential for IP3-evoked 45Ca2+ release. Two nonhydrolyzable ATP analogues satisfied the nucleotide requirement for IP3-evoked 45Ca2+ release. IP3 strongly stimulated 45Ca2+ efflux at low temperatures (1 to 15 degrees C). Decreasing the temperature from 37 to 4 degrees C inhibited the rate of IP3-stimulated efflux by only about 33%. The failure of such low temperatures to strongly inhibit IP3-induced 45Ca2+ efflux suggests that IP3 activated a Ca2+ channel, rather than a carrier, by a ligand-binding, rather than a metabolic, reaction.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.