Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Forskolin and Phorbol Myristate Acetate Inhibit Intracellular Ca2+ Mobilization Induced by Amitriptyline and Bradykinin in Rat Frontocortical Neurons

Abstract— Regulations of the increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) and inositol 1, 4, 5-trisphosphate (IP₃) production by increasing intracellular cyclic AMP (cAMP) levels or activating protein kinase C (PKC) were studied in rat frontocortical cultured neurons. Amitriptyline (AMI; 1 mM), a trìcyclic antidepressant, and bradykinin (BK; 1 μM) stimulated IP₃ production and caused transient [Ca²⁺]_i increases. Pretreatment with forskolin (100mkUM, 15 min) decreased the AMI-and BK-induced [Ca²⁺]_i increases by 33 and 48%, respectively. However, this treatment had no effect on the AMI-and BK-induced IP₃ productions. Dibutyryl-cAMP (2 mM, 15 min) also decreased the AMI-and BK-induced [Ca²⁺]ⁱ increases by 23 and 47%, respectively. H-8 (30 μM), an inhibitor of protein kinase A (PKA), attenuated the ability of forskolin to inhibit the AMI-and BK-induced [Ca²⁺]_i increases, suggesting that the activation of cAMP/PKA was involved in these inhibitory effects of forskolin. On the other hand, forskolin treatment had no effect on 20 mM caffeine-, 10 μM glutamate-, or 50 mM K⁺-induced [Ca²⁺]_i increases. Pretreatment with phorbol 12-myristate 13-acetate (PMA; 100 nM, 90 min) decreased both the AMI-induced [Ca²⁺]_i increases and the IP₃ production by 31 and 25%, respectively. H-7 (200 μM), an inhibitor of PKC, inhibited the ability of PMA to attenuate the [Ca²⁺]_i increases. PMA also inhibited the BK-induced IP₃ production and the [Ca²⁺]_i increases. Taken together, these results suggest that activation of cAMP/ PKA may inhibit the IP₃-mediated Ca²⁺ release from internal stores; on the other hand, activation of PKC may inhibit the phosphatidylinositol 4,5-bisphosphate breakdown and consequently reduce the [Ca²⁺]_i increases or inhibit independently both responses. PKA and PKC may differently regulate the phosphatidylinositol-Ca²⁺ signaling in rat frontocortical cultured neurons.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

Background

Diminished calcium (Ca²⁺) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.

Methodology/Principal Findings

VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP₃R) and the SR Ca²⁺ pumps (SERCA 2 and 3). Generally, a decrease in IP₃R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP₃R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca²⁺ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca²⁺ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca²⁺ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca²⁺ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca²⁺ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.

Conclusions/Significance

This work demonstrates that the previously-reported reduced Ca²⁺ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP₃R Ca²⁺ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Functional characterization of a muscarinic receptor in the smooth muscle of the shark (Squalus acanthias) ventral aorta

A suite of muscarinic receptor blockers was used to characterize the receptor(s) mediating the contractile effect of acetylcholine (ACh) on isolated rings of ventral aorta from the dogfish shark, Squalus acanthias. The M₂/M₄-specific inhibitor N,N’-bis(6-{[(2-methoxyphenyl) methyl] amino} hexyl) -1,8- octane diamine tetrahydrochloride (methoctramine) did not reduce the efficacy of ACh, and the M₃-specific inhibitor 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displaced the ACh concentration-response curve to the right at much lower concentrations than the M₁-specific inhibitor (5-11-dihydro-11- [4-methyl-l-piperazinyl)acetyl] -6H-pyrido[2,3-b] [1,4] benzodiazepin-6-one dihydrochloride) (pirenzepine). It appears, therefore, that an M₃-type muscarinic receptor is expressed in the aortic vascular smooth muscle of the dogfish shark.     

Glucocorticoid amplifies vasopressin-induced phosphoinositide hydrolysis in aortic smooth muscle cells

It has been reported that glucocorticoid modifies phosphoinositide (PI) hydrolysis stimulated by vasoactive agents in vascular smooth muscle cells. In the present study, we investigated the point at which glucocorticoid affects vasopressin-induced PI hydrolysis in primary cultured rat aortic smooth muscle cells. The pretreatment with dexamethasone significantly amplified the formation of inositol trisphosphate (IP₃) induced by vasopressin in a dose-dependent manner in a range of 1 pM to 10 nM. The effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone had little effect on the number of vasopressin receptor and its affinity to vasopressin. The pretreatment with dexamethasone also amplified the formation of IP₃ induced by NaF, a GTP-binding protein activator, or angiotensin II. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, significantly reduced the dexamethasone-induced enhancement of IP₃ formation stimulated by vasopressin, angiotensin II or NaF. 4α-Phorbol-12, 13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the enhancement by dexamethasone. These results strongly suggest that glucocorticoid amplifies vasopressin-induced PI hydrolysis at a point downstream from GTP-binding protein in primary cultured rat aortic smooth muscle cells, and that the activation of PKC has a negative feedback effect on the amplification by glucocorticoid of vasopressin-induced PI hydrolysis.     

Protein kinase C activator phorbol 12, 13-dibutyrate inhibits platelet activating factor-stimulated Ca2+ mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells

The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBa) dose-dependently inhibited platelet-activating factor (PAF)-induced [Ca²⁺]_i elevation and inositol monophosphate (IP₁) accumulation in neurohybrid NG108-15 cells with IC₅₀ values of 162 nM and 35 nM, respectively. Pretreatment of NG108-15 cells with PKC inhibitor H-7 partially prevented the inhibitory effect of PDBu on PAF-induced [Ca²⁺]_i elevation as well as PI metabolism in NG108-15 cells. Pretreatment of the cells with pertussis toxin (PTX) resulted in a dose-dependent inhibition of PAF-induced IP₁ and IP₃ accumulation but only slightly affected PAF-induced [Ca²⁺]_i elevation in NG108-15 cells. The results reveal that PAF receptor-mediated Ca²⁺ mobilization and PI metabolism in NG108-15 cells are regulated by PKC while a PTX-sensitive G protein is coupled to PAF receptor for inducing activation of phospholipase C.     

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca²⁺ release induced with 0.18 μM IP₃ is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP₃, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca²⁺ uptake into intracellular stores, does not influence IP₃ induced Ca²⁺ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP₃ to the same cellular preparations was investigated. We found that binding of IP₃ to a high affinity receptor site (K_d approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP₃ binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP₃ with the same affinity (K_d approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP₃ was displaced from these subcellular membranes with similar kinetics by unlabelled IP₃ and decavanadate. The data suggest that the inhibitory action of decavanadate on IP₃ induced Ca²⁺ release is a consequence of its effect on binding of IP₃ to its receptor.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular IP3-sensitive Ca2+ pools

Abstract. A comparative study of inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca²⁺]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP, in a dose-dependent manner. However, when stimulated by various concentrations of IP₃ (1–10 μM), the increase in [Ca²⁺]i of BRCP was always two- to threefold higher than that in BRPE. Subcellular-fractionation studies showed that a single population of IP₃ binding site with a high affinity and high specificity of IP₃ mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [³²P]-IP₃ binding sites (Kd 1.9–2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP-dependent Ca²uptake and IP₃-dependent Ca²⁺ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca²⁺ whereas BRPE only released 0.07 nmol Ca²⁺ with the same dose of IP₃ (5 μM). The selectively higher density of IP, binding sites in coupling to the larger Ca²⁺-release in the membrane of BRCP suggests that the quantity of Ca²⁺ mobilized is determined by the spatially preferential distribution of membrane-associated IP₃ binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP,-induced DNA replication.     

Intracellular signalling of epinephrine in rat hepatocytes during fetal development and hepatic regeneration

The changes in intracellular calcium concentration and IP₃ production after the addition of epinephrine were analysed in adult, fetal (20th–22nd day of intrauterine life), and regenerating rat hepatocytes (4 h–24 h after partial hepatectomy) to determine whether the signal transduction is the same in quiescent proliferating and differentiating cells.The epinephrine treatment causes a significative cytosolic calcium transient in hepatocytes isolated in the last day of fetal life (22-day old) and in the early stage of regeneration (4 h). This effect is not significant in the previous stage of fetal life (20-day old) and at the onset of M phase of cell cycle after partial hepatectomy (24 h).[³H]myo inositol incorporation into IP₃ and IP₄ is higher in 20 day fetal and regenerating hepatocytes with respect to the control. In these cells the epinephrine does not affect basal level of IP₃ and IP₄, while it causes a substantial increase of these inositol phosphates in adult hepatocytes.[³H]myo inositol incorporation into PIP₂ is very low at the 20th day of fetal life. Epinephrine has no effect on this parameter in fetal and regenerating hepatocytes.Our results show that the epinephrine signal is mediated differently in proliferating and in quiescent hepatocytes.     

Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Ca-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP₃) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP₃ mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP₃ mass levels under resting conditions. The IP₃ mass was neither modified by changes in external Ca²⁺ nor by a Ca²⁺-free medium containing EGTA. IP₃ mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K⁺ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca²⁺ emphasizing that Ca²⁺ entry, independently of the influx mechanism involved, leads to an IP₃ increase. The phospholipase C_β inhibitors U-73122 and U-73343 reduced K⁺-stimulated IP₃ levels while K⁺-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP₃ by agents that stimulate ACh release may suggest a possible link between IP₃ metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca²⁺]_i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca²⁺]_i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min⁻¹). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.     

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris-phosphate (IP₃), intracellular calcium, [Ca²⁺]_i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP₃ and a rapid increase in [Ca²⁺]_i in cells loaded with the Ca²⁺-responsive fluorescent dye fura-2. The prompt AII-induced [Ca²⁺]_i spike was not affected by incubating HCEC in Ca²⁺-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca²⁺ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca²⁺ from intracellular stores. The AII-triggered increases in IP₃, [Ca²⁺]_i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar¹, Val⁵, Ala⁸-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca²⁺]_i and independent of PKC. © 1996 Wiley-Liss, Inc.     

Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca(2+) increase associated with the ATP-induced increase in ciliary beat frequency

An increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP₃) receptor-dependent Ca²⁺ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP₃ peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca²⁺ from internal stores and an influx of extracellular Ca²⁺, respectively. In addition, ATP-dependent Ca²⁺ mobilization required protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP₃ receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca²⁺]_i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca²⁺ influx is also dependent on IP₃ receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP₃ receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP₃ receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP₃ receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP₃ receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP₃ receptor can directly trigger Ca²⁺ influx in response to ATP. transduction mechanisms; P2Y receptor; calcium influx     

All-or-none shortening of α-toxin-permeabilized single smooth muscle cells from guinea-pig taenia caecum and acetylcholine-induced Ca2+ sensitization

Isolated single smooth muscle cells from guinea-pig taenia caecum, which responded to acetylcholine (ACh) in an all-or-none manner, were permeabilized by exposure to Staphylococcus aureus α-toxin. Permeabilized cells responded to 0.3 μM or 0.6 μM Ca²⁺ with maximal shortening, showing an all-or-none response. The addition of ACh resulted in a decrease in the concentration of Ca²⁺ required to trigger a threshold response. GDP inhibited, and treatment with GTP mimicked the action of ACh. The addition of inositol 1,4,5-trisphosphate (IP₃) in the permeabilized cells caused a slight cell shortening. These results suggest that α-toxin-permeabilized cells retain their receptor function and show augmentation of Ca²⁺ sensitivity by stimulation of the muscarinic receptor or GTP-binding protein(s). Moreover, it is suggested that the intact cells show an all-or-none response to ACh when the intracellular Ca²⁺ level is greater than the threshold concentration due to signaling being coupled to muscarinic receptor stimulation. This then causes the augmentation of Ca²⁺ sensitivity and reaches the threshold to evoke Ca²⁺ influx and IP₃-induced Ca²⁺ release.     

Melatonin Receptor-Mediated Stimulation of Phosphoinositide Breakdown in Chick Brain Slices

Abstract: The direct effect of melatonin and related agonists on Li⁺-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-³H]-inositol. The melatonin receptor agonist 6-chloromelatonin (10–100 µM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 µM) was rapid as transient increases in IP₃/IP₄ (maximal increase, 29% at 20 s) and IP₂ levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP₁ level (30% at 5 min), when the amount of IP₃/IP₄ and IP₂ had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 µM) in isolated myo-[2-³H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5′-O-(3-thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration-dependent manner the IP₁ accumulation stimulated by 6-chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5-hydroxytryptamine (10 µM)-, but not 6-chloromelatonin (10 µM)-, induced IP₁ accumulation. Melatonin receptor agonists increased IP₁ accumulation in a concentration-dependent manner reaching different maximal responses. N-Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP₁ accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5-hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.