Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the rabbit, calcitonin has been shown to enhance calcium (Ca2+) reabsorption in the early distal tubule. The aim of the present study was to investigate the mechanism of this action, using isolated luminal and basolateral membranes of distal tubules. The tubule suspensions were preincubated in the presence or absence of 10(-7) M calcitonin. The luminal or basolateral membranes were subsequently purified and 45Ca transport through the vesicles was measured using the rapid filtration technique. Results were compared with those obtained from proximal tubule membranes. In the proximal tubules, calcitonin had no effect on Ca2+ uptake by luminal membranes. In the distal tubules, the presence of Na+ in the incubation medium strongly decreased the uptake of Ca2+ by luminal membranes. Preincubation of distal tubules with calcitonin partially restored this uptake. We previously reported a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. To determine through which messenger(s) calcitonin enhances Ca2+ transport by the distal tubules, we first confirmed that the hormone stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Therefore, we studied the actions of cAMP and phorbol 12-myristate 13 acetate (PMA) on Ca2+ transport by the distal luminal membranes. Incubation of distal tubule suspensions with dibutyryl cAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. We conclude that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms: the opening of low affinity Ca2+ channels in the luminal membrane and the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.
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PMID:Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron. 918 93

1. Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5-HT) in response to increased external Ca2+. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled receptor for external Ca2+ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release. 2. Whole-cell recordings in the permeabilized-patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+]0) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca2+]0 activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near -25 mV indicating poor ion selectivity. 3. The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd3+ > Ba2+ > Ca2+ > > Mg2+. The insensitivity of these cells to high external Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4. Elevation of [Ca2+]0 also stimulates increases in intracellular Ca2+ concentration ([Ca2+]i) and this effect is largely inhibited by the Ca2+ channel blocker nimodipine, indicating that L-type voltage-gated Ca2+ channels contribute to the response to elevated [Ca2+]0. 5. Elevated [Ca2+]0 induces an inward current under conditions where the only permeant external cation is Ca2+, indicating that influx via the cation conductance is another source of the increases in [Ca2+]i. 6. Extracellular Ca2+ stimulates 5-HT release with an EC50 of 1.5 mM. Nimodipine blocks 90% of the Ca2+0-induced 5-HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca2+ channels in CaR-induced hormone secretion. Although earlier studies indicate that high [Ca2+]0 induces release of Ca2+ from intracellular stores, thapsigargin-induced depletion of these stores did not affect secretion from these cells, indicating that Ca2+ influx is necessary and sufficient for the Ca2+0-induced 5-HT secretion. 7. Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca2+0-induced 5-HT release by 50% while phorobol ester-induced 5-HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway. 8. We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 had no effect on the response to elevated [Ca2+]0. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca(2+)-induced 5-HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca2+]0.
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PMID:Mechanism of extracellular Ca2+ receptor-stimulated hormone release from sheep thyroid parafollicular cells. 923 95

We previously reported that parathyroid hormone and calcitonin increase Ca2+ uptake by purified distal luminal membranes. This effect is mimicked by high concentrations of cAMP. However, both hormones stimulate adenylate cyclase and phospholipase C. The purpose of the present study was to investigate the role of the phospholipase C pathway in the hormone action, and the interrelationship between the two messengers. Distal tubules from rabbit kidneys were incubated with dibutyryl cAMP (dbcAMP) or PMA, or both, and Ca2+ uptake by purified luminal membranes was measured by the rapid filtration technique. Incubation of the distal tubules with 1 mM dbcAMP significantly increased Ca2+ transport by the luminal membranes. A dose-response curve showed a half-maximal stimulation with 0.82 mM dbcAMP. In contrast, treatment of the tubules with 10 nM, 100 nM or 1 microM PMA did not influence Ca2+ uptake by these membranes. However, the addition of 100 nM PMA to low concentrations of dbcAMP strongly increased this uptake. The presence of cAMP or protein kinase C inhibitors prevented the effects of either a high concentration of dbcAMP alone or a low concentration of dbcAMP combined with 100 nM PMA. Our laboratory has already reported that Ca2+ uptake by the distal luminal membranes displays two-component kinetics. dbcAMP increased the Vmax of the low-affinity component, whereas a combination of the two messengers stimulated the Vmax of both the low- and high-affinity components. From these results, we conclude that: (1) in the distal tubule cells, activation of both protein kinases A and C is necessary for the stimulation of Ca2+ transport by the luminal membrane; (2) the combined effect of protein kinases A and C involves both components of the Ca2+-transport kinetics.
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PMID:Ca2+ transport by the luminal membrane of the distal nephron: action and interaction of protein kinases A and C. 937 90

1. D-Myo inositol 1,2,6 trisphosphate (alpha-trinositol, pp56), an isomer of the second messenger substance, inositol 1,4,5 trisphosphate, has an interesting pharmacological profile that includes antagonism of a number of neuropeptide Y (NPY)-mediated cellular processes. The ability of pp56 to inhibit selectively the myocardial contraction mediated by NPY in relation to the responses to other cardioactive peptides, including endothelin-1, calcitonin gene-related peptide (CGRP), secretin and vasoactive intestinal peptide (VIP), was assessed. In order to investigate the possible interaction of pp56 with mechanisms of inositol phosphate signalling generated in heart muscle cells by activation of the beta-isoenzyme of phospholipase C (PLC beta), noradrenaline was used as a positive control, and isoprenaline and forskolin were included as negative controls. 2. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM). The concentrations of agonists used were in the region of their maximally effective concentrations for myocyte contraction and the concentration of pp56 was in the range of 1-100 microM. Contractile activity was monitored by video microscopy and maximum shortening determined by image analysis. 3. In the absence of agonist, contractile amplitudes following 20 min preincubation with pp56 were not different from that observed in the absence of pp56. Pp56 (1-100 microM) inhibited significantly the positive contractile response to noradrenaline (5 microM) in the presence of propranolol (500 nM), such that the response was almost completely attenuated at the highest concentration of the inhibitor. Pp56 did not inhibit the positive contractile responses to forskolin (40 microM) or isoprenaline (100 nM). 4. NPY alone does not influence the basal level of contraction of cardiomyocytes, but can attenuate isoprenaline-stimulated contraction and can increase contractile amplitude from basal when the transient outward current is blocked with 4-aminopyridine. In the presence of isoprenaline (100 nM), the negative response to NPY (100 nM) was attenuated significantly by pp56 (1-100 microM). With 4-aminopyridine, the positive contractile response to NPY (200 nM) was decreased by pp56, although this was not statistically significant. 5. Pp56 inhibited the positive contractile responses to CGRP (1 nM) and endothelin-1 (20 nM) completely, but did not affect the responses to secretin (20 nM) or VIP (20 nM). 6. In conclusion, these data challenge the previously obtained selectivity of pp56 as an antagonist of NPY-mediated cellular processes, since responses to CGRP and endothelin-1 were at least equally sensitive. Furthermore, as pp56 discriminated clearly in its inhibition of responses to alpha-adrenoceptor by comparison with beta-adrenoceptor/adenylate cyclase stimulation, it appears that pp56 may be a useful pharmacological agent with which to distinguish between PLC beta-dependent and PLC beta-independent coupling mechanisms. On this basis, further evidence has been obtained that, in rat cardiomyocytes, the contractile responses to NPY, CGRP and endothelin-1 are attributable to the activation of PLC beta-dependent pathways, whereas the responses to secretin and VIP are mediated by PLC beta-independent pathways.
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PMID:Use of D-myo inositol 1,2,6 trisphosphate to inhibit contractile activity in rat ventricular cardiomyocytes induced by neuropeptide Y and other cardioactive peptides through phospholipase C. 942 11

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.
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PMID:Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor. 964 99

While it is well established that adenylyl cyclase and phospholipase C-beta are two proximal signal effectors for the calcitonin receptor, the more distal signaling pathways are less well characterized. G protein-coupled receptors can activate Erk1/2 by Gs-, Gi-, or Gq-dependent signaling pathways, depending on the specific receptor and cell type examined. Since the calcitonin receptor can couple to all three of these G proteins, the ability of calcitonin to activate Erk1/2 was investigated. Calcitonin induced time- and concentration-dependent increases in Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 phosphorylation and activation in a HEK 293 cell line that stably expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin, which inactivates Gi, and calphostin C, a protein kinase C inhibitor, each partially inhibited calcitonin-induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In contrast, neither forskolin nor H89, a protein kinase A inhibitor, had a significant effect on basal or calcitonin-stimulated Erk1/2 phosphorylation. Our results suggest that the calcitonin receptor induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by parallel Gi- and PKC-dependent mechanisms. The calcitonin-induced elevation of cytosolic free Ca2+ was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca2+ by chelating both cytosolic and extracellular Ca2+ abolished the response. However, the change in Ca2+ that is induced by calcitonin is not sufficient to account for the calcitonin-induced Erk1/2 phosphorylation, since treatment with 100 nM ionomycin or 10 microM thapsigargin, each of which induced elevations of Ca2+ comparable to those induced by calcitonin, induced significantly less Erk1/2 phosphorylation than that induced by calcitonin. Erk1/2 may have important roles as downstream effectors mediating cellular responses to calcitonin stimulation.
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PMID:The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium. 967 14

Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether beta gamma subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-beta isoforms (beta1, beta3, beta4, but not beta2) and G-proteins (Galphaq/11 and Galphas). Only phospholipase C-beta1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-gamma have no effect. Activation of PLC-beta1 by CGRP involves the Galphaq/11 subunit, which is insensitive to pertussis toxin, but not Gbeta gamma subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.
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PMID:Activation of phospholipase C-beta1 via Galphaq/11 during calcium mobilization by calcitonin gene-related peptide. 968 62

Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.
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PMID:Calcitonin gene-related peptide (CGRP) increases intracellular free Ca2+ concentrations but not cyclic AMP formation in CGRP receptor-positive osteosarcoma cells (OHS-4). 1020 67

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.
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PMID:Protein kinase C antagonizes pertussis-toxin-sensitive coupling of the calcitonin receptor to adenylyl cyclase. 1023 69

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.
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PMID:3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity. 1038 24


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