When S1P meets ATP

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels

Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.     

Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

Transient receptor potential vanilloid 4 (TRPV4) channels are Ca²⁺-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca²⁺ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca²⁺ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca²⁺ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type Ca_V1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive G_q-coupled receptor (opto-α₁AR) resulted in TRPV4-mediated Ca²⁺ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca²⁺ influx into arterial myocytes.     

G-Protein Regulation of an L-Type Calcium Channel Current in Canine Jejunal Circular Smooth Muscle

Calcium entry into smooth muscle cells is essential to maintain contractility. In canine jejunal circular smooth muscle cells the predominant calcium entry pathway is through L-type calcium channels. The aim of this study was to determine the G-protein regulation of L-type calcium channel current (I _CaL) in isolated canine jejunal circular smooth muscle cells. Barium (80 mm) was used as the charge carrier. GTP-γS and GTP increased maximal inward current from 118.7 ± 12 pA to 227.5 ± 21.5 pA (n= 8) and 174.6 ± 10.1 pA (n= 6) respectively. The increase in inward current was blocked by nifedipine suggesting it was through L-type calcium channels. Pertussis toxin did not alter baseline I _CaL while cholera toxin increased I _CaL from 125 ± 19 pA in controls (n= 6) to 347 ± 30 pA (n= 4). Staurosporine inhibited the increase in current evoked by GTP-γS and calyculin further increased I _CaL over the increase evoked by GTP-γS. The results suggest that cholera toxin sensitive G-proteins activate L-type calcium channels in isolated canine jejunal circular smooth muscle cells through protein phosphorylation. Received: 27 March 1997/Revised: 3 July 1997     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Differential regulation of calcium channel coding genes by prolonged depolarization

Calcium channels must be subjected to a very precise regulation in order to preserve cell function and viability. Voltage gated calcium channels (VGCC) represent the main pathway for calcium entry in excitable cells. This explains why depolarization induces a rapid-onset and short-term inactivation of calcium currents. Contrarily to this well-documented mechanism to maintain calcium below toxic levels, the regulatory pathways inducing longer-lasting changes and cell surface expression of functional calcium channels are largely unknown. Since calcium is a main player in the activity-dependent regulation of many genes, we hypothesize that calcium channel coding genes could be also subjected to activity-dependent regulation. We have used prolonged depolarization to analyze the effects of sustained intracellular calcium elevation on the mRNAs coding for the different alpha(1) pore-forming subunits of the calcium channels expressed in chromaffin cells. Our findings reveal that persistent depolarization is accompanied by a prolonged intracellular calcium elevation and reduction of calcium current. This calcium current inhibition could be mediated, at least partially, by the downregulation of the mRNAs coding for several alpha(1) subunits. Thus, we show here that depolarization inhibits the expression of Ca(V)1.1, Ca(V)1.2, Ca(V)1.3, Ca(V)2.2 and Ca(V)2.3 mRNAs, while the Ca(V)2.1 mRNA remains unmodified. Moreover, such downregulation of channels depends on calcium entry through the L-type calcium channel, as both mRNA and calcium current changes induced by depolarization are abrogated by L-type channel specific blockers.     

L-type calcium channel β subunit modulates angiotensin II responses in cardiomyocytes

Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Ca(v)β subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Ca(v)β subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Ca(v)β subunit isoform, with Ca(v)β(1b) containing channels being more strongly regulated. Ca(v)β(2a) containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Ca(v)β subunit-dependent manner. These data demonstrate that Ca(v)β subunits alter the magnitude of inhibition of L-type current by Angiotensin II.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone

The smooth muscle cells of resistance arteries depolarize and contract when intravascular pressure is elevated. This is a central characteristic of myogenic tone, which plays an important role in regulation of blood flow in many vascular beds. Pressure-induced vascular smooth muscle depolarization depends in part on the activation of cation channels. Here, we show that activation of these smooth muscle cation channels and pressure-induced depolarization are mediated by protein kinase C in cerebral resistance arteries. Diacylglycerol, phorbol myristate acetate, and cell swelling activate a cation current that we have previously shown is mediated by transient receptor potential channels. These currents, as well as the smooth muscle cell depolarizations of intact arteries induced by diacylglycerol, phorbol ester, and elevation of intravascular pressure, are nearly eliminated by protein kinase C inhibitors. These results suggest a major mechanism of myogenic tone involves mechanotransduction through phospholipase C, diacylglycerol production, and protein kinase C activation, which increase cation channel activity. The associated depolarization activates L-type calcium channels, leading to increased intracellular calcium and vasoconstriction.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.     

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells

OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

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.     

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.