Rise In Cytosolic Free Ca2 Research Articles

Endothelin mobilizes calcium and enhances glucose uptake in cultured human skeletal myoblasts and L6 myotubes.

In this study we used endothelin as a paradigm to explore the concept that some vasoactive agents, acting through mobilization of Ca2+ and stimulation of protein kinase C, can interact with human skeletal muscle and modify its glucose transport. Cultured human skeletal myoblasts from the vastus lateralis demonstrated two subclasses of high-affinity endothelin receptors and a robust increase in cytosolic free Ca2+ upon exposure to endothelin. The endothelin-evoked rise in cytosolic free Ca2+ primarily resulted from Ca2+ mobilization from intracellular organelles. Both endothelin and insulin enhanced [3H]deoxy-D-glucose uptake in human myoblasts, but their effects were not additive. These findings also were observed in differentiated myotubes of L6 skeletal muscle cells. Moreover, [3H]deoxy-D-glucose uptake in human myoblasts was enhanced by treatment with phorbol 12-myristate 13-acetate. The endothelin- and insulin-mediated increases in [3H]deoxy-D-glucose were totally ablated by treatment with calphostin C. Such observations suggest that endothelin can enhance glucose uptake in human skeletal muscle. This is mediated through mechanisms that are at least partially protein kinase C dependent. Thus, increased levels of endothelin in vascular beds may contribute to altered glucose metabolism in essential hypertension.

Ca2+]i and protein kinase C in vasopressin-induced prostacyclin and ANP release in rat cardiomyocytes.

Exposure of cultured, spontaneously beating rat cardiomyocytes to arginine vasopressin (AVP) led to marked increases in the release of prostacyclin (PGI2) and atrial natriuretic peptide (ANP). These responses were accompanied by a rapid, transient rise of cytosolic free Ca2+ concentration ([Ca2+]i) and of membranous protein kinase C (PKC) activity. Ca2+ influx and PKC activity appeared to play important but distinct roles in AVP-induced cellular responses, insofar as only AVP-induced ANP secretion was abolished by the Ca2+ channel antagonist nifedipine, whereas both AVP-induced PGI2 production and ANP release were abolished by the PKC inhibitors staurosporine and CGP-41251. The AVP-induced increase in [Ca2+]i could also be mimicked with the vasopressin (V1-subtype) agonist Octapressin, but not with the V2-agonist 1-desamino-8-D-arginine vasopressin, and was fully abolished by the V1-antagonist [d(CH2)5Tyr(Me)]AVP, but not by d(CH2)5-D-Leu-VAVP (V1-/V2-antagonist). These results indicate that V1-vasopressinergic receptors mediate AVP-induced PGI2 production and ANP secretion in rat cardiomyocytes and that, whereas both Ca2+ influx and PKC activation are required for AVP-induced ANP secretion, AVP-induced PGI2 formation is mainly regulated by PKC.

Increased intracellular Na+ augments mobilization of Ca2+ from SR in vascular smooth muscle cells.

The effect of a rise in intracellular Na+ concentration ([Na+]cyt) on the amount of Ca2+ in intracellular stores was studied in vascular smooth muscle cells from the A7r5 line. The relative amount of stored Ca2+ was estimated in fura 2-loaded cells by the rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) evoked by Ca2+ release from the sarcoplasmic reticulum (SR). To improve the detection of released Ca2+, extrusion of Ca2+ from the cytosol was minimized by using nominally Na+/Ca(2+)-free medium containing 0.5 mM La3+ [for vasoconstrictor experiments, the medium contained 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and no La3+]. Ca2+ release was triggered by thapsigargin (TG), an SR Ca(2+)-ATPase inhibitor, and by the vasoconstrictors arginine vasopressin (AVP) and serotonin (5-HT). Incubation with 1-3 mM ouabain for 20 min, which raises [Na+]cyt from 4.4 to 9.0 mM, increased "resting" [Ca2+]cyt only slightly (from 87 to 122 nM). However, ouabain greatly augmented the release of Ca2+ evoked by TG [from 639 nM (control) to 1,021 nM], by AVP (from 993 to 1,597 nM), and by 5-HT (from 559 to 1,486 nM). Ouabain-induced augmentation of TG-evoked Ca2+ release was not affected by 10 microM verapamil; this implies that the effect of ouabain was not due to Ca2+ entry through voltage-gated Ca2+ channels. The response to TG was not augmented when ouabain was applied for 20 min in Na(+)-free medium (Na+ replaced by equimolar N-methyl-D-glucamine) to prevent [Na+]cyt from rising.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets.

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.

Thapsigargin inhibits voltage-activated calcium channels in adrenal glomerulosa cells

Thapsigargin, an inhibitor of the microsomal Ca2+ pumps, has been extensively used to study the intracellular Ca2+ pool participating in the generation of the agonist-induced Ca2+ signal in various cell types. A dual effect of this agent was observed in bovine adrenal zona glomerulosa cells. At nanomolar concentrations, thapsigargin stimulated a sustained Ca2+ influx, probably resulting from Ca(2+)-store depletion. In contrast, when added at micromolar concentrations, thapsigargin prevented the rise in cytosolic free Ca2+ concentration ([Ca2+]c) induced by K+. This inhibitory effect of thapsigargin on voltage-activated Ca2+ channels was confirmed by measuring Ba2+ currents by the patch-clamp technique. Both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels were affected by micromolar concentrations of thapsigargin. Analysis of the current-voltage relationship for T-type channels revealed that thapsigargin did not modify the sensitivity of these channels to the voltage, but decreased the maximal current flowing through the channels. In conclusion, thapsigargin appears to exert a dual effect on adrenal glomerulosa cells. At lower concentrations, this agent induces a sustained Ca2+ entry, whereas at higher concentrations it decreases [Ca2+]c by blocking voltage-activated Ca2+ channels.

Plasminogen activation stimulates an increase in intracellular calcium in human synovial fibroblasts.

Both plasminogen (Pg) and urinary-type Pg activator (u-PA), but not tissue-type Pg activator (t-PA), bind to normal and rheumatoid arthritis (RA) human synovial fibroblasts in culture with high affinity and in a dose-dependent manner. Single cell intracellular Ca2+ responses to Pg and u-PA were studied using Fura-2 and digital imaging fluorescence microscopy. Pg activation by u-PA on the surface of RA synovial fibroblasts induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 90 s. Pg kringle 4 and the alpha 2,3-linked sialic acid in the carbohydrate chain bound to Thr245 are involved in mediating the increases in [Ca2+]i. This response is not observed in normal synovial fibroblasts, suggesting that RA synovial fibroblasts have altered responses to the binding and activation of Pg on their surfaces.

Ca2+ and Zn2+ Dependence of DNA Synthesis in Untransformed and in Ha-rasval-12-Expressing NIH 3T3 Cells

The role of Ca2+ and Zn2+ in the initiation of DNA synthesis in NIH 3T3 fibroblasts and c-Ha-rasval-12 oncoprotein-expressing (NIH 3T3) cells has been studied. Entrapment of the Ca2+ chelator, BAPTA (30 μM), into the cells totally blocked a serum-induced rise in cytosolic free Ca2+ ([Ca2+]i) as determined with fura-2. Serum starvation for 24 h considerably reduced DNA synthesis in control NIH 3T3 fibroblasts. BAPTA treatment reduced serum-induced DNA synthesis and totally inhibited platelet-derived growth factor-induced DNA synthesis in these cells. DNA synthesis of the c-Ha-rasval-12-expressing fibroblasts was little affected by serum starvation and unaffected by entrapment of BAPTA into the cells. Intracellular Zn2+ was measured using the fluorescent probe TSQ in intact cells. As determined using image analysis the TSQ fluorescence was distributed throughout the cytoplasm and concentrated around the nucleus. The permeable Zn2+ chelator, TPEN, at a concentration of 10 μM, caused a maximal reduction in TSQ-available Zn2+. This concentration of TPEN totally blocked DNA synthesis both in control and c-Ha-rasval-12-expressing fibroblasts. Upon addition of 11 μM Zn2+ DNA synthesis was restored even after TPEN addition. [3H]Thymidine incorporation itself was also sensitive to TPEN treatment. The results suggest that c-Ha-rasval-12-induced proliferation is independent of changes in [Ca2+]i. A specific role of Zn2+ in c-Ha-ras-induced proliferation is unlikely since ras-expressing and control cells reacted similarly to Zn2+ deprivation. There seems to be a constant requirement for the presence of Zn2+ in cell proliferation.

Intracellular Ca2+ sequestration and release in intact bovine retinal rod outer segments. Role in inactivation of Na-Ca+K exchange

Intracellular Ca2+ sequestration and Ca2+ release was analyzed in intact rod outer segments (ROS) purified from bovine retinas. Ca2+ influx in Ca(2+)-depleted and fully bleached ROS was mediated exclusively by the Na-Ca+K exchanger and was measured both as a rise in cytosolic free Ca2+ with fluo 3 and as a total transmembrane Ca2+ flux with 45Ca. Ca2+ fluxes across the ROS plasma membrane were not completely reversible, in small part due to inactivation of the Ca2+ extrusion mode of the Na-Ca+K exchanger but mostly due to sequestration of cytosolic Ca2+ into the intradiskal space. Ca2+ release from the intradiskal space into the cytosol could be induced by low concentrations of the Ca2+ ionophore A23187 which gave rise to increases in cytosolic free Ca2+ by several hundred nanomolar (low A23187 concentrations did not affect the Ca2+ permeability of the ROS plasma membrane). We have used intracellular Ca2+ release induced by A23187 to examine inactivation of the rapid Ca2+ efflux mode mediated by the plasma membrane Na-Ca+K exchanger. Inactivation of Na-Ca+K exchange appeared to be dependent on intradiskal Ca2+ as it was abolished by selective Ca2+ permeabilization of the disk membrane by A23187. We discuss possible physiological roles for Ca2+ sequestration and release from ROS disks.

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells.

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca2+ ([Ca2+]i) in C6 glioma cells with an EC50 of 60 +/- 4 and 10 +/- 5 microM, respectively. The threshold concentration of ATP (3 microM) for increasing [Ca2+]i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca2+ and Na+ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca2+ channels (Co2+, Mn2+, La3+, or Cd2+). In Ca(2+)-free medium, ATP caused only a transient increase in [Ca2+]i as opposed to a sustained [Ca2+]i increase in normal medium. The ATP-induced elevation of [Ca2+]i was resistant to Na+ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attenuated by La3+. The differences in the characteristics of ATP-caused P1 hydrolysis and [Ca2+]i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca2+ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P1-purinergic receptors, the C6 glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na+]o dependency for ATP-induced PI turnover require further investigation.

Thapsigargin‐evoked changes in human platelet Ca2+, Na+, pH and membrane potential.

1. In this work we explored the effect of thapsigarin on the intracellular Ca2+, pH, Na+ and membrane potential in human platelets. These parameters were monitored using the fluorescent probes fura-2, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, sodium-binding benzofuran isophthalate, and 3,3'-dipropylthiadicarbocyanine iodide. 2. Thapsigargin caused an increase in the cytosolic Ca2+, coupled with cytosolic alkalinization. Thapsigargin-induced alkalinization was Na(+)-dependent, indicating that thapsigargin stimulated the Na(+)-H+ exchange. 3. Using Mn2+ as a Ca2+ surrogate, we showed that thapsigargin activated Ca2+ channels at relatively low levels of cytosolic Ca2+, suggesting that a rise in cytosolic free Ca2+ is not the signal for the activation of these channels. 4. Thapsigargin-induced increase in the cytosolic free Ca2+ was greater in Na(+)-containing medium than in Na(+)-free medium, suggesting that Na(+)-dependent mechanisms participate in the regulation of platelet cytosolic Ca2+. 5. Thapsigargin not only increased the cytosolic Ca2+, but also elevated the cytosolic free Na+. The latter effect was more pronounced in Ca(2+)-free medium, a finding that may indicate that some of the Na+ enters through Ca2+ entry pathways. 6. Finally, thapsigargin evoked sustained platelet hyperpolarization which was attenuated by charybdotoxin, indicating thapsigargin-induced stimulation of Ca(2+)-sensitive K+ channels. 7. Together these observations demonstrate a multifactorial effect of thapsigargin on platelets that can be utilized to further understand platelet ionic homeostasis.

Tumor necrosis factor induces enhanced responses to platelet‐activating factor and differentiation in human monocytic Mono Mac 6 cells

Human Mono Mac 6 cells exhibit characteristics of mature blood monocytes. Treatment of these cells with human recombinant human tumor necrosis factor (TNF) resulted in an increase in phagocytosis and phorbol myristate acetate-stimulated superoxide anion production at 12 h and growth retardation occurring at 24 h. Moreover, TNF induced a moderate increase of CD14 surface antigen expression, used as a phenotypic marker of monocyte/macrophage differentiation. Platelet-activating factor (PAF) stimulated a rapid rise in cytosolic free Ca++ ([Ca++]i) of 308 +/- 93 nM in TNF-treated cells compared to untreated cells (33 +/- 8 nM, n = 4). The effect of TNF was dose and time dependent, evident after 12 h and maximal at 48 h. The enhanced PAF-induced [Ca++]i rise was inhibited by the PAF receptor antagonist L-659,989 and EGTA, indicating receptor-dependent Ca++ influx. Furthermore, L-659,989 and PAF inhibited specific 3H-labeled PAF binding in TNF-treated, but not in untreated cells. Consistently, PAF stimulated arachidonic acid release only in TNF-treated cells. Preincubation of cells with anti-TNF monoclonal antibodies abolished TNF-induced effects, but failed to block lipopolysaccharide (LPS) effects. Distinct mechanisms of action by LPS were reflected by the different ability to induce surface antigen expression. In conclusion, the enhancement of PAF responses by TNF, associated with functional characteristics of differentiation in Mono Mac 6 cells, may represent a specific mechanism of cooperative interaction between PAF and TNF in inflammation, sepsis, immunoregulation and atherogenesis.

Divergent Effects of Acute and Chronic Ethanol Exposure on Contraction and Ca2+ Mobilization in Cultured Vascular Smooth Muscle Cells

In cultured rat vascular smooth muscle cells (VSMC), acute preincubation of 100 mmol/L ethanol for 30 min attenuated the number of contracting cells in response to (10(-7) mol/L) arginine vasopressin (AVP) (P < .01). In contrast, VSMC cultured chronically for 3 days in medium supplemented with 100 mmol/L ethanol enhanced (10(-7) mol/L) AVP-induced shape change (P < .01). Specific 3H-AVP binding to VSMC after acute or chronic exposure to 100 mmol/L ethanol did not differ from those of control experiments. Acute ethanol pretreatment attenuated basal, 10(-7) mol/L AVP-, 65 mmol/L K(+)-stimulated Ca2+ uptake, in a dose-dependent manner. In contrast, 100 mmol/L ethanol for 4 days enhanced the (P < .001) AVP- 10(-7) mol/L and (P < .01) 65 mmol/L K(+)-stimulated 45Ca2+ uptake. Acute ethanol exposure inhibited and chronic ethanol administration enhanced Ca2+ uptake stimulated by 6 x 10(-7) mol/L Bay K 8644, an activator of voltage-sensitive Ca2+ channels. Nifedipine, a blocker of these Ca2+ channels, diminished AVP-stimulated (P < .02) and K(+)-induced (P < .001) Ca2+ uptake more potently in VSMC pretreated for 4 days with 100 mmol/L ethanol than in control cells. Acute ethanol preexposure for 30 min attenuated AVP-stimulated inositol trisphosphate (IP3) formation (P < .05) and the rise in cytosolic free Ca2+ ([Ca2+]i) (P < .01). In contrast, chronic ethanol-treated VSMC enhanced IP3 formation (P < .05) and the rise in [Ca2+]i (P < .01) in response to AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human tumor necrosis factor alpha induces calcium oscillation and calcium-activated chloride current in human neutrophils. The role of calcium/calmodulin-dependent protein kinase.

The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigated whether TNF induces cytosolic free Ca2+ changes and Ca(2+)-activated Cl- current, respectively. Bath application of 1000 units/ml recombinant human TNF alpha (rhTNF alpha) induced a rise in cytosolic free Ca2+ in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNF alpha activated Cl- current in 80% of tested cells; the activated current was blocked by 10 microM 5-nitro-2-3-phenylpropylamino)benzoic acid, a Cl- channel blocker. The current was similarly activated by 1 microM ionomycin, a Ca2+ ionophore. To study the mechanism by which rhTNF alpha induced Ca(2+)-activated Cl- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (5 mM), the calmodulin antagonist (2 microM), CaM kinase II-(290-309), or the inhibitory peptide (10 microM), CaM kinase II-(273-302), the current was no longer activated by rhTNF alpha. The intracellular application of the control peptide (10 microM), CaM kinase II-(284-302), or the protein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu27]PKC-(19-36), peptide (5 microM) did not block the rhTNF alpha-induced Cl- current. These results show that Ca2+ changes are associated with the effects of rhTNF alpha and that CaM kinase plays a role in the mechanism underlying rhTNF alpha-induced activation of Ca(2+)-activated Cl- current in human neutrophils.

Oscillations of cytosolic free calcium concentration in the presence of intracellular antibodies to phosphatidylinositol 4,5-bisphosphate in voltage-clamped guinea-pig hepatocytes.

In liver cells, the stimulation of alpha 1-adrenoceptors by noradrenaline induces the production of Ins(1,4,5)P3 through the degradation of membrane polyphosphoinositides [PtdIns(4,5)P2]. InsP3 evokes in turn the release of Ca2+ from internal stores. Our results show that the internal perfusion of single guinea-pig hepatocytes with monoclonal anti-PtdInsP2 antibody blocks the rise in cytosolic free Ca2+ concn. ([Ca2+]i) evoked by noradrenaline, an InsP3-dependent agonist, but not by the monohydroxylated bile acid taurolithocholate 3-sulphate, which is known to permeabilize the endoplasmic reticulum. In these conditions, the bile acid elicited either fast or slow fluctuations of [Ca2+]i independently of any InsP3 production. The responses to the bile acid were also observed in the absence of external Ca2+. The presence of intracellular anti-PtdInsP2 antibody does not affect the response to a photolytic release of InsP3 (1.5 microM final concn.) from a caged precursor.

Intracellular free [Ca 2+] and [Na +] in response to capsaicin in cultured dorsal root ganglion cells

Intracellular free Ca 2+ and Na + concentrations in cultured rat dorsal root ganglion cells were studied using the intracellular fluorescent probes fura-2 and SBFI, respectively. Capsaicin increased both intracellular [Ca 2+] and [Na +]. A rise in cytosolic free Ca 2+ could still be seen if Na + was removed from the outer medium. The results suggest that capsaicin opens up ion channels which are permeable both to Ca 2+ and Na +.

Acute variations in extracellular pH modulate transduction pathways of PTH in rat proximal tubule.

An increase in circulating parathyroid hormone (PTH) has been shown to enhance the capacity for the kidney to excrete an acid as well an alkaline load, which suggests that changes in systemic acid-base status may modulate the effect of the hormone on bicarbonate absorption in proximal tubule. In the present study, we tested the possibility that acute variations in extracellular pH (pHe), obtained by modifying bicarbonate concentration at constant PCO2 (40 mmHg), may modulate the responses of intracellular messengers coupled to PTH receptors in a preparation of freshly isolated proximal tubule fragments. Variations in pHe, which induced parallel variations in intracellular pH (pHi), did not affect unstimulated values for adenosine 3',5'-cyclic monophosphate (cAMP) production, inositol trisphosphate accumulation, or cytosolic free Ca2+ concentration. In contrast, reducing pHe from 7.4 to 7.2 elicited a decrease of the PTH-induced cAMP production, whereas increasing pHe from 7.4 to 7.6 enhanced it. The ability for cholera toxin and forskolin (which both bypass PTH receptors) to stimulate cAMP formation was diminished at pHe 7.2 and enhanced at pHe 7.6 (the increase did not achieve statistical significance in the presence of forskolin), suggesting that variations in pHe and/or pHi may affect per se adenylyl cyclase activity. Conversely, reducing pHe from 7.4 to 7.2 enhanced the PTH-induced inositol trisphosphate accumulation and rise in cytosolic free Ca2+ whereas increasing pHe from 7.4 to 7.6 had opposite effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.

A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.

The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans.

Sodium azide (NaN3), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10-15 min) application followed by removal of 3 mM NaN3 results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3-5 mM). In addition, application of NaN3, even at very low [Ca2+]o, reversibly increases cytosolic Ca2+ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca2+, by itself, is not sufficient to trigger insulin secretion.