Proteinase-activated receptor-1 and -2 induce the release of chemokine GRO/CINC-1 from rat astrocytes via differential activation of JNK isoforms, evoking multiple protective pathways in brain

Activation of both PAR-1 (proteinase-activated receptor-1) and PAR-2 resulted in release of the chemokine GRO (growth-regulated oncogene)/CINC-1 (cytokine-induced neutrophil chemoattractant-1), a functional counterpart of human interleukin-8, from rat astrocytes. Here, we investigate whether the two PAR receptor subtypes can signal separately. PAR-2-induced GRO/CINC-1 release was independent of protein kinase C, phosphoinositide 3-kinase and MEK (mitogen-activated protein kinase kinase)-1/2 activation, whereas these three kinases were involved in PAR-1-induced GRO/CINC-1 release. Despite such clear differences between PAR-1 and PAR-2 signalling pathways, JNK (c-Jun N-terminal kinase) was identified in both signalling pathways to play a pivotal role. By isoform-specific loss-of-function studies using small interfering RNA against JNK1-3, we demonstrate that different JNK isoforms mediated GRO/CINC-1 secretion, when it was induced by either PAR-1 or PAR-2 activation. JNK2 and JNK3 isoforms were both activated by PAR-1 and essential for chemokine GRO/CINC-1 secretion, whereas PAR-1-mediated JNK1 activation was mainly responsible for c-Jun phosphorylation, which was not involved in GRO/CINC-1 release. In contrast, PAR-2-induced JNK1 activation, which failed to phosphorylate c-Jun, uniquely contributed to GRO/CINC-1 release. Therefore our results show for the first time that JNK-mediated chemokine GRO/CINC-1 release occurred in a JNK isoform-dependent fashion and invoked PAR subtype-specific mechanisms. Furthermore, here we demonstrate that activation of PAR-2, as well as PAR-1, rescued astrocytes from ceramide-induced apoptosis via regulating chemokine GRO/CINC-1 release. Taken together, our results suggest that PAR-1 and PAR-2 have overlapping functions, but can activate separate pathways under certain pathological conditions to rescue neural cells from cell death. This provides new functional insights into PAR/JNK signalling and the protective actions of PARs in brain.     

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.     

Storage and release of ATP from astrocytes in culture

ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.     

Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells

Background

This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs).

Methods

The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 μg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs.

Results

Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 μg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059), p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion.

Conclusions

Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.

     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

The release of slow-reacting substance of anaphylaxis from guinea pig lung: effects of calcium antagonists

The effects of three calcium antagonists, verapamil, lanthanum, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were studied on the release of slow-reacting substance of anaphylaxis (SRS-A) from ovalbumin-sensitized chopped guinea pig lung parenchyma in calcium-containing and calcium-free media. The SRS-A levels (mean +/- SEM) obtained from tissues incubated in normal and calcium-free Krebs-bicarbonate buffer were 51 +/- 8 (N = 19) and 21 +/- 4 (N = 14) U/mL, respectively. TMB-8 (0.1-10 microM), a reported intracellular calcium antagonist, reduced antigen-stimulated SRS-A release from lung tissue incubated in calcium-containing, but not calcium-free, medium; A23187-induced SRS-A release from normal guinea pig lung was not significantly altered by TMB-8 at concentrations up to 10 microM. Verapamil and lanthanum consistently reduced SRS-A release only at high concentrations (100 microM and 1mM, respectively). The quantities of SRS-A released from lung tissue incubated in the presence of verapamil in normal medium were similar to those obtained in calcium-free medium. Tissues incubated in the presence of potassium chloride (60 and 100 mM) did not release significant quantities of SRS-A, and release which did occur was not blocked by verapamil, suggesting that antigen-induced SRS-A release is not dependent on membrane depolarization and that verapamil was not exerting inhibition via blockade of voltage-dependent calcium channels. These data suggest that although intracellular calcium is important for the regulation of SRS-A secretion from guinea pig lung tissue, extracellular calcium is necessary for optimal release of SRS-A.     

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of prolactin stimulated pig oocytes

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of pig oocytes activated by prolactin was investigated, using the fluorescent dye chlortetracycline. In the presence of extracellular calcium, the inhibitor of protein kinase C Ro 31-8220 increased calcium exit from intracellular stores in pig oocytes after prolactin treatment. In calcium-free medium, Ro 31-8220 exerted effect on calcium release from intracellular stores. In calcium-free medium, prolactin did not stimulate calcium release from intracellular stores of oocytes in the presence of thimerosal, while in the presence of protein kinase C inhibitor, prolactin increased Ca2+ content from intracellular stores in such oocytes. These data suggest a direct involvement of protein kinase C in the processes of regulation of Ca2+ exit from intracellular stores of pig oocytes stimulated by prolactin.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Effects of TMB-8 and dantrolene on ACTH- and angiotensin-induced steroidogenesis by frog interrenal gland: evidence for a role of intracellular calcium in angiotensin action

The influence of intracellular calcium on the steroidogenic response of adrenocortical tissue to ACTH and angiotensin has been studied in the frog, using a perifusion system technique. The release of corticosterone, aldosterone and prostaglandins in the effluent medium was monitored by specific radioimmunoassays. TMB-8 and dantrolene, two potential blockers of calcium mobilization from intracellular pool(s), were tested. Dantrolene (5 X 10(-5) M) significantly reduced basal and angiotensin-induced corticosterone and aldosterone production but had little effect on ACTH-evoked steroid release. Conversely TMB-8 (10(-4) M) profoundly depressed spontaneous as well as ACTH- and angiotensin II-induced corticosteroid secretion, suggesting that this compound may affect not only calcium mobilization from the endoplasmic reticulum pool but also calcium influx. Adrenal glands perifused with both dantrolene and calcium-free medium showed no response to angiotensin II. Conversely, in calcium-free conditions and in the presence of dantrolene, angiotensin II still caused an increase in prostaglandin synthesis. Taken together, these results indicate that 1) dantrolene is a more specific agent than TMB-8 in inhibiting calcium mobilization from intracellular pool(s); 2) ACTH increases corticosteroidogenesis without inducing mobilization of intracellular calcium; 3) angiotensin II stimulates both the efflux of calcium from the endoplasmic reticulum and the influx of calcium through the plasma membrane; 4) calcium is required after prostaglandin production in the steroidogenic response of frog interrenal gland to angiotensin II.     

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ～30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-β-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.     

Role of calcium in the secretion of leptin from white adipocytes

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.     

Identification of a common receptor for three types of rat cytokine-induced neutrophil chemoattractants (CINCs)

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/macrophage inflammatory protein-2 (MIP-2), members of the CXC chemokine family, are potent chemotactic factors for neutrophils. In order to identify the receptor for CINCs, rat CXC chemokine receptor 2 (CXCR2) was cloned and expressed in HEK293 cells. CINC-1, CINC-2 and CINC-3 induced calcium mobilizations dose-dependently in CXCR2-transfected cells, whereas formyl-methionyl-leucyl-phenylalanine (FMLP) did not. CINC-3 induced enhancement of cytoplasmic calcium concentration more potently than CINC-1 and CINC-2, and desensitized calcium transients induced by CINC-1 and CINC-2, which were essentially identical to those observed in rat neutrophils. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of three types of CINCs almost completely. The mutant CINC-3, whose amino-terminal amino acid sequence (SELR) was replaced to AAR, lost chemotactic activity of its own but inhibited that of CINC-1 and CINC-2 potently, and that of CINC-3 weakly. The results indicate that rat CXCR2 on neutrophils is the unique receptor for all three types of CINCs, and CINC-1/-2 and CINC-3 exert different biological activities through the common receptor.     

Calcium dependence of phenylephrine-, endothelin-, and potassium chloride-stimulated atrial natriuretic factor secretion from long term primary neonatal rat atrial cardiocytes

Since calcium is involved in both excitation-secretion and excitation-contraction coupling, it was of interest to evaluate its involvement in atrial natriuretic factor (ANF) release from atrial cardiocytes. In medium containing physiological levels of calcium (1.4 mM), the secretion of ANF from primary atrial cells was stimulated from 3- to 6-fold by a variety of agents including KCl, phenylephrine, and endothelium (ET). However, in medium containing 2 nM calcium, KCl was incapable of increasing ANF secretion above basal levels, while the stimulatory effects of phenylephrine and ET were only partially diminished. Nifedipine or verapamil could mimic the effects of the 2 nM calcium medium on KCl-, phenylephrine-, and ET-stimulated ANF secretion. Kinetic studies indicated that during the initial 5 min of ET-stimulated secretion the cells exhibited little requirement for extracellular calcium; however, the requirement was more apparent during the sustained secretion observed between 10 min and 2 h of secretagogue exposure. Additionally, the stimulation of ANF secretion by ET increased to a maximum of about 15-fold over basal by 10-min after ET application; subsequent to this time there was an apparent functional desensitization wherein the rate of secretion decreased by approximately 3-4-fold and remained at this level for the duration of secretagogue exposure up to 2 h. All forms of stimulated secretion could be inhibited through ionomycin-mediated depletion of intracellular calcium pools. Taken together, these results indicate that atrial cardiocytes require both extracellular and intracellular calcium to support maximal rates of stimulated ANF secretion, and that intracellular calcium pools may be used during the early phase of secretion, while the extracellular source of calcium may be important for the sustained phase of secretion.     

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.     

The influence of exogenous cyclic nucleotides and theophylline on the hydrokinetic and ecbolic function of the isolated perfused canine pancreas (author's transl)

The influence of exogenous cyclic nucleotides or theophylline either on basal or stimulated volume and protein secretion is studied on the isolated perfused canine pancreas in dependence on varied extracellular calcium concentrations. Bt2cAMP or theophylline do not influence basal secretory rates of pancreatic juice but potentiate secretin-stimulated volume output. They additionally increase basal protein secretion under exclusive secretin stimulation and potentiate dose-dependently CCK- or acetylcholine-induced protein output. The hydrokinetic and ecbolic effects of Bt2cAMP and theophylline persist in a calcium-free medium but fail in normalizing inhibited protein secretion during calcium deprivation. Bt2cGMP neither increases basal nor stimulated volume and protein secretion. The demonstrated influence of Bt2cAMP and theophylline on ductal volume and acinar protein secretion accomplishes two criteria, as suggested by Sutherland, for cAMP as second messenger for secretin and CCK or acetylcholine as well.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases

The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase (MMP-12) protein. Active serine protease is responsible for induction, as demonstrated by the absence of MMP-12 induction in plasminogen(Plg)-treated urokinase-type plasminogen activator-deficient macrophages. Since increased MMP-12 protein secretion was not accompanied by an increase in MMP-12 mRNA, we examined post-translational mechanisms. Protein synthesis was not required for early release of MMP-12 but was required for later secretion of activated enzyme. Immunofluorescent microscopy demonstrated basal expression in macrophages that increased following serine proteinase exposure. Inhibition of MMP-12 secretion by hirudin and pertussis toxin demonstrated a role for the thrombin G protein-coupled receptor (protease-activated receptor 1 (PAR-1)). PAR-1-activating peptides were able to induce MMP-12 release. Investigation of signal transduction pathways involved in this response demonstrate the requirement for protein kinase C, but not tyrosine kinase, activity. These data demonstrate that plasmin and thrombin regulate MMP-12 activity through distinct mechanisms: post-translational secretion of preformed MMP-12 protein, induction of protein secretion that is protein kinase C-mediated, and extracellular enzyme activation. Most importantly, we show that serine proteinase MMP-12 regulation in macrophages occurs via the protein kinase C-activating G protein-coupled receptor PAR-1.     

Dual regulation of leptin secretion: intracellular energy and calcium dependence of regulated pathway

Rodent leptin is secreted by adipocytes and acutely regulates appetite and chronically regulates body weight. Mechanisms for leptin secretion in cultured adipocytes were investigated. Acutely, energy-producing substrates stimulated leptin secretion about twofold. Biologically inert carbohydrates failed to stimulate leptin secretion, and depletion of intracellular energy inhibited leptin release. There appears to be a correlation between intracellular ATP concentration and the rate of leptin secretion. Insulin increased leptin secretion by an additional 25%. Acute leptin secretion is calcium dependent. When incubated in the absence of calcium or in the presence of intracellular calcium chelators, glucose plus insulin failed to stimulate leptin secretion. In contrast, basal leptin secretion is secreted spontaneously and is calcium independent. Adipocytes from fatter animals secrete more leptin, even in the absence of calcium, compared with cells from thinner animals. Acute stimulus-secretion coupling mechanisms were then investigated. The potassium channel activator diazoxide and the nonspecific calcium channel blockers nickel and cadmium inhibited acute leptin secretion. These studies demonstrate that intracellular energy production is important for acute leptin secretion and that potassium and calcium flux may play roles in coupling intracellular energy production to leptin secretion.