The relationship between alpha 1-adrenergic receptor occupation and the mobilization of intracellular calcium

We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact BC3H-1 muscle cells in order to examine the relationship between the number of surface receptors occupied and the functional response. [3H]Prazosin has been used to measure receptor number as well as the binding kinetics with surface receptors, and the observed equilibrium and kinetic constants are in close accord with values obtained previously in cellular homogenates. Since alpha 1-agonist-elicited 45Ca2+ efflux can be monitored over intervals of 3 min or less and prazosin dissociation from its receptor has a t 1/2 of 44 min, prazosin can be employed to produce a pseudoirreversible inactivation of receptors. A comparison of the remaining receptors and residual response reveals an inverse linear relationship between receptors inactivated by prazosin and 45Ca2+ efflux. A similar result is obtained following fractional receptor inactivation with the irreversible alkylating agent phenoxybenzamine. Parameters of receptor occupation and response also correlate well for the agonist phenylephrine and for the competitive antagonist phentolamine. The unitary relationship between sites available for occupation and response indicates that the alpha 1 receptor does not function as an oligomer where fewer bound antagonist molecules are required to block the receptor than sites of agonist occupation necessary for activation. Moreover, substantial evidence has accrued in intact smooth muscle for a receptor reserve or nonlinear coupling between alpha 1 receptor occupation and contraction in smooth muscle. Our findings demonstrate that such behavior does not exist for alpha 1 receptor-elicited mobilization of Ca2+ in the BC3H-1 muscle cell.     

Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers

The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1-adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores.     

Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

Dopamine D2 receptor modulation of carotid body type 1 cell intracellular calcium in developing rats

Carotid chemoreceptor type 1 cells release dopamine, which inhibits carotid chemoreceptor activity via dopamine D2 autoreceptors on type 1 cells. Postnatal changes in dopaminergic modulation may be involved in postnatal chemoreceptor development. The present study explores dopaminergic modulation of the intracellular calcium ([Ca(2+)](i)) response to hypoxia in type 1 cells from 1, 3, and 11- to 16-day-old rats. Using fura-2, we studied the effects of quinpirole, a D2 receptor agonist, on type 1 cell [Ca(2+)](i) response to 90-s hypoxia challenges (Po(2) approximately 1-2 mmHg). Cells were sequentially exposed to the following challenges: 1) hypoxia control, 2) hypoxia plus quinpirole, and 3) hypoxia plus quinpirole plus sulpiride (D2 receptor antagonist). In the 11- to 16-day-old group, type 1 cell [Ca(2+)](i) increased approximately 3 to 4-fold over resting [Ca(2+)](i) in response to hypoxia. Quinpirole (10 microM) significantly blunted the peak [Ca(2+)](i) response to hypoxia. Repeat challenge with hypoxia plus 10 microM quinpirole in the presence of 10 microM sulpiride partially restored the hypoxia [Ca(2+)](i) response. In sharp contrast to the older aged group, 10 microM quinpirole had minimal effect on hypoxia response of type 1 cells from 1-day-olds and a small but significant effect at 3 days of age. We conclude that stimulation of dopamine D2 receptors inhibits type 1 cell [Ca(2+)](i) response to hypoxia, consistent with an inhibitory autoreceptor role. These findings suggest dopamine-mediated inhibition and oxygen sensitivity increase with age on a similar time course and do not support a role for dopamine as a major mediator of carotid chemoreceptor resetting.     

Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.     

Differences in intracellular calcium mobilization by interferon-beta and interferon-gamma in RPMI-4788 cells

Human interferon (IFN) stimulates a 1.5- to 1.7-fold transient increase in the concentration of cytoplasmic-free calcium ion ([Ca2+]i) within 10-20 s upon exposure of RPMI-4788 cells to IFN. This early event of IFN-induced [Ca2+]i mobilization was measurable by loading the cells with Fura-2AM, a fluorescent Ca2+ indicator. The mobilization induced by IFN-beta or IFN-gamma was dependent on the concentration of each IFN. The increased [Ca2+]i gradually returned to its resting level within 60 s. The addition of EGTA (0.5-10 mM) to medium induced a marked decrease in the amount of [Ca2+]i mobilized by IFN-beta and a partial decrease by IFN-gamma. This finding suggests that the mechanisms of [Ca2+]i mobilization by IFN-beta and IFN-gamma might be different. While IFN-beta-induced mobilization may be mainly from an influx of the extracellular calcium ion ([Ca2+]o), IFN-gamma-induced mobilization may be a summation of an influx of [Ca2+]o and a release from intracellular Ca2+ stores.     

Fast potentiation of glycine receptor channels of intracellular calcium in neurons and transfected cells

Inhibitory glycine receptors (GlyRs) are mainly expressed in the spinal cord and in the midbrain, where they control motor and sensory pathways. We describe here a fast potentiation of GlyR by intracellular Ca2+. This phenomenon was observed in rat spinal cord neurons and in transfected human cell lines. Potentiation develops in <100 ms, is proportional to Ca2+ influx, and is characterized by an increase in GlyR apparent affinity for glycine. Phosphorylation and G protein pathways appear not to be involved in the potentiation mechanism. Single-channel recordings in cell-attached and excised patches, as well as whole-cell data suggest the presence of a diffusible cytoplasmic factor that modulates the GlyR channel gating properties. Ca2+-induced potentiation may be important for rapid modulation of glycinergic synapses.     

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.     

Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.     

Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.     

Direct relationship between intracellular calcium mobilization and phospholipase D activation in prostaglandin E-stimulated human erythroleukemia cells.

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.     

Changes in the intracellular calcium concentration upon activation of rat superior cervical ganglion neurons

Changes in the intracellular calcium concentration induced by activation of neurons of the isolated intact rat superior cervical ganglion were recorded. It is concluded that stimulation within the physiological range of frequencies can effectively increase the intracellular calcium concentration in these neurons. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 400–402, July–October, 2007.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

Nuclear localization of functional metabotropic glutamate receptor mGlu1 in HEK293 cells and cortical neurons: role in nuclear calcium mobilization and development

The Group I metabotropic glutamate receptor (mGlu1) plays an important role in neuromodulation, development, and synaptic plasticity. Using immunocytochemistry, subcellular fractionation, and western blot analysis, the present study shows that mGlu1a receptors are present on nuclear membranes in stably transfected human embryonic kidney 293 (HEK293) cells as well as being endogenously expressed on rat cortical nuclei. Both glutamate and the group I agonist, quisqualate, directly activate nuclear mGlu1 receptors leading to a characteristic oscillatory pattern of calcium flux in isolated HEK nuclei and a slow rise to plateau in isolated cortical nuclei. In either case calcium responses could be terminated upon application of the mGlu1-selective antagonist, 7-(hydroxyamino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. Responses could also be blocked by ryanodine and inositol 1,4,5-triphosphate receptor inhibitors, demonstrating the involvement of these calcium channels. Agonist activation of intracellular receptors was driven by Na(+)-dependent and -independent processes in nuclei isolated from either HEK or cortical neurons. Finally, mGlu1 nuclear receptors were dramatically up-regulated in the course of post-natal development. Therefore, like the other Group I receptor, mGlu5, mGlu1 can function as an intracellular receptor, suggesting a more encompassing role for nuclear G protein-coupled receptors and downstream signaling elements in the regulation of nuclear events.     

Effect of tetracaine on thrombocyte aggregation and mobilization of the intracellular calcium

Tetracaine (1 mM), a local anesthetics, lowers a degree of aggregation of human thrombocytes which is induced by thrombin (0.15 u/ml) and suppresses its appearance. Aggregation of thrombocytes induced by phorbol ester, TPA (10-8 M), an activator of protein kinase C, is inhibited completely by the mentioned doses of the anesthetics. In the presence of tetracaine the release of intracellular Ca is lower to some extent, but then it surpasses the control level. It is established that under the action of ionophore A23187 tetracaine exerts no effect on mobilization of intracellular Ca2+.     

Arachidonic acid induces mobilization of calcium stores and c-jun gene expression: evidence that intracellular calcium release is associated with c-jun activation.

Arachidonic acid (AA) plays a signaling role in the induction of several genes. We previously demonstrated that AA induces c-jun gene expression in the stromal cell line +/+.1 LDA 11 by a signaling pathway involving activation of the c-jun amino-terminal kinase (JNK). This study investigated the role of calcium in AA signaling of c-jun activation in +/+.1 LDA 11 cells. AA (10-50 microM) caused a rapid dose-dependent rise in cytosolic calcium. AA-induced calcium mobilization involved both influx of extracellular calcium and the release of intracellular calcium. The importance of calcium was investigated by variation of the extracellular calcium concentration, chelation of intracellular calcium and by calcium ionophore-induced influx of extracellular calcium. AA-induced c-jun gene expression and increased luciferase activity of a construct containing the high affinity AP-1 binding site was decreased in cells preincubated with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)-eThane-N,N,N',N',-tetraacetic acid tetra(aceToxymethyl-esTer) (BAPTA-AM, 10 microM) prior to stimulation with AA. Similarly, chelation of intracellular calcium decreased AA-induced JNK activation. On the contrary, changes in the extracellular calcium concentration had no effect. Also, ionophore A23187 failed to induce c-jun and JNK activation either alone than in combination with AA. These results suggested that calcium was required for AA-dependent activation of c-jun, but that calcium alone was insufficient to induce activation of c-jun. Thus, release of calcium from intracellular stores is implicated in the signaling pathway of AA-induced c-jun activation in stromal cells.