Protease-activated receptor-2 increases exocytosis via multiple signal transduction pathways in pancreatic duct epithelial cells

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.     

Characterization of the protease-activated receptor-1-mediated contraction and relaxation in the rat duodenal smooth muscle

Activation of protease-activated receptor-1 (PAR-1) produces a dual action, apamin-sensitive relaxation followed by contraction, in the rat duodenal smooth muscle, which is partially dependent on activation of L-type Ca2+ channels, protein kinase C (PKC) or tyrosine kinase (TK), and resistant to tetrodotoxin. The present study further characterized the PAR-1-mediated duodenal responses. Removal of extracellular Ca2+ as well as SK&F96365 reduced the contraction due to the PAR-1 agonist TFLLR-NH2 (TFp-NH2) by 60-80% that was similar to the extent of the inhibition by nifedipine. Lowering of the extracellular Na+ concentration, but not IAA-94, a Cl- channel inhibitor, reduced both the PAR-1-mediated contraction and relaxation by about 50%. U73122, a phospholipase C (PLC) inhibitor, or wortmannin, a phosphatidyl inositol 3'-kinase (PI3K) inhibitor, significantly reduced the PAR-1-mediated contraction, but not the relaxation, by itself, as the PKC inhibitor GF109203X and the TK inhibitor genistein did. U73122 or wortmannin, like GF109203X, when applied in combination with genistein, significantly reduced the PAR-1-mediated relaxation. The relaxation was resistant to antagonists of PACAP receptors, VIP receptors and P2 purinoceptors. Thus, the PAR-1-mediated contraction is considered to be dependent on intracellular and extracellular Ca2+, the influx of the latter being induced through activation of L-type Ca2+ channels triggered by the enhanced Na+ permeability, and that PLC and PI3K, in addition to PKC and TK, are involved in the PAR-1-mediated dual responses. Furthermore, non-adrenergic, non-cholinergic nerve neurotransmitter candidates that may modulate K+ channels do not appear to contribute to the relaxation by PAR-1 activation.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Modulation of cardiac Ca2+ channel by Gq-activating neurotransmitters reconstituted in Xenopus oocytes

L-type dihydropyridine-sensitive voltage dependent Ca(2+) channels (L-VDCCs; alpha(1C)) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, was G(q)- and PLC-independent and Ca(2+)-dependent, but not Ca(2+)-mediated, as only basal levels of Ca(2+) were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations.     

Protein kinase C-dependent and -independent inhibition of Ca(2+) influx by phorbol ester in rat pancreatic beta-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic beta-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca(2+) concentration ([Ca(2+)](i)) elevated by the glucose stimulation when tested in isolated rat beta-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized beta-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca(2+) currents through L-type Ca(2+) channels. BIM prevented the PMA inhibition of the Ca(2+) currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic beta-cells, defeating its own inhibition of the Ca(2+) influx through L-type Ca(2+) channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.     

Mechanisms of light adaptation in Drosophila photoreceptors

Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels. Ca(2+) influx via these channels is required for light adaptation, but although several molecular targets of Ca(2+)-dependent feedback have been identified, their contribution to adaptation is unclear. By manipulating cytosolic Ca(2+) via the Na(+)/Ca(2+) exchange equilibrium, we found that Ca(2+) inhibited the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca(2+) levels (0.1-10 microM Ca(2+)) and accurately mimicked light adaptation. However, PLC activity monitored with genetically targeted PIP(2)-sensitive ion channels (Kir2.1) was first inhibited by much higher (>/= approximately 50 microM) Ca(2+) levels, which occur only transiently in vivo. Ca(2+)-dependent inhibition of PLC, but not the LIC, was impaired in mutants (inaC) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca(2+)-dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca(2+) levels, whereas rapid inhibition of PLC by local Ca(2+) transients is required to terminate the response and ensures that PIP(2) reserves are not depleted during stimulation.     

Three distinct Ca(2+) influx pathways couple acetylcholine receptor activation to catecholamine secretion from PC12 cells

Amperometry and microfluorimetry were employed to investigate the Ca(2+)-dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine-evoked exocytosis was entirely dependent on extracellular Ca(2+) but was only partly blocked by Cd(2+), a nonselective blocker of voltage-gated Ca(2+) channels. Secretion and rises of [Ca(2+)](i) observed in response to nicotine could be almost completely blocked by methyllycaconitine and alpha-bungarotoxin, indicating that such release was mediated by receptors composed of alpha7 nicotinic acetylcholine receptor subunits. Secretion and [Ca(2+)](i) rises could also be fully blocked by co-application of Cd(2+) and Zn(2+). Release evoked by muscarine was also fully dependent on extracellular Ca(2+). Muscarinic receptor activation stimulated release of Ca(2+) from a caffeine-sensitive intracellular store, and release from this store induced capacitative Ca(2+) entry that could be blocked by La(3+) and Zn(2+). This Ca(2+) entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca(2+)](i) and exocytosis via Ca(2+) influx through voltage-gated Ca(2+) channels, alpha7 subunit-containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca(2+) entry.     

Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways.

Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca(2+) from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca(2+) signaling pathway activated by 10-100 pM AVP. This pathway is distinguished from the PLC pathway by its dependence on protein kinase C (PKC) and L-type voltage-sensitive Ca(2+) channels (VSCC). In the present study, we used isolated, pressurized rat mesenteric arteries to examine the contributions of these different Ca(2+) signaling mechanisms to AVP-induced vasoconstriction. AVP (10(-14)-10(-6) M) induced a concentration-dependent constriction of arteries that was reversible with a V(1a) vasopressin receptor antagonist. Half-maximal vasoconstriction at 30 pM AVP was prevented by blockade of VSCC with verapamil (10 microM) or by PKC inhibition with calphostin-C (250 nM) or Ro-31-8220 (1 microM). In contrast, acute vasoconstriction induced by 10 nM AVP (maximal) was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min, the remaining vasoconstriction induced by 10 nM AVP was partially dependent on PKC activation and almost fully dependent on VSCC. These results suggest that different Ca(2+) signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca(2+) signaling pathway that is dependent on PKC activation and can be inhibited by Ca(2+) channel blockers.     

Decreased function of voltage-gated potassium channels contributes to augmented myogenic tone of uterine arteries in late pregnancy

Increased pressure-induced (myogenic) tone in small uteroplacental arteries from late pregnant (LP) rats has been previously observed. In this study, we hypothesized that this response may result from a diminished activity of vascular smooth muscle cell (SMC) voltage-gated delayed-rectifier K(+) (K(v)) channels, leading to membrane depolarization, augmented Ca(2+) influx, and vasoconstriction (tone). Elevation of intraluminal pressure from 10 to 60 and 100 mmHg resulted in a marked, diltiazem-sensitive rise in SMC cytosolic Ca(2+) concentration ([Ca(2+)](i)) associated with a vasoconstriction of uteroplacental arteries of LP rats. In contrast, these changes were significantly diminished in uterine arteries from nonpregnant (NP) rats. Gestational augmentation of pressure-induced Ca(2+) influx through L-type Ca(2+) channels was associated with an enhanced SMC depolarization, the appearance of electrical and [Ca(2+)](i) oscillatory activities, and vasomotion. Exposure of vessels from NP animals to 4-aminopyridine, which inhibits the activity of K(v) channels, mimicked the effects of pregnancy by increasing pressure-induced depolarization, elevation of [Ca(2+)](i), and development of myogenic tone. Furthermore, currents through K(v) channels were significantly reduced in myocytes dissociated from arteries of LP rats compared with those of NP controls. Based on these results, we conclude that decreased K(v) channel activity contributes importantly to enhanced pressure-induced depolarization, Ca(2+) entry, and increase in myogenic tone present in uteroplacental arteries from LP rats.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.     

Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation

Pressure-induced decreases in arterial diameter are accompanied by membrane depolarization and Ca(2+) entry via voltage-gated Ca(2+) channels. Recent evidence also suggests the involvement of Ca(2+) sensitization of the contractile proteins. Both PKC and Rho kinase are candidate second messengers for the mediation of the sensitization process. We investigated the signaling pathways of pressure-induced decreases in rat cerebral artery diameter in vessels that were depolarized with a 60 mM potassium-physiological salt solution (KPSS). Arteries were mounted on a pressure myograph, and pressure-induced constrictions were recorded. In some experiments simultaneous changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were recorded by using fura 2 fluorescence photometry. Pressure increases induced constriction with significant changes in [Ca(2+)](i) at high pressures (60-100 mmHg). The ratio of the change in diameter to change in [Ca(2+)](i) was greater for pressure-induced constriction compared with constriction produced by depolarization with 60 mM KPSS, suggesting that in addition to increases in [Ca(2+)](i), enhanced myofilament Ca(2+) sensitivity occurs during pressure-induced decreases in arterial diameter. Depolarizing the membrane with 60 mM KPSS increased [Ca(2+)](i) via a Ca(2+) influx pathway insensitive to PKC inhibition. Cerebral arteries were able to maintain their diameters in the continued presence of 60 mM KPSS. Pressure-induced constriction under these conditions was not associated with further increases in Ca(2+) but was abolished by selective inhibitors of PLC, PKC, and Rho kinase. We report for the first time that in rat cerebral arteries, pressure-induced decreases in arterial diameter are not only due to increases in voltage-gated Ca(2+) influx but also to accompanying increases in myofilament sensitivity to Ca(2+) mediated by PKC/Rho kinase activation.     

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (Ca2+L) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca2+L channels and lead to Ca2+ entry and vasoconstriction.     

Beta- and alpha-adrenergic cross-signaling for L-type Ca current is impaired in transgenic mice with constitutive activation of epsilonPKC

It is well established that beta-adrenoceptor stimulation activates PKA and alpha(1)-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of alpha(1)-adrenoceptors inhibits beta-adrenoceptor stimulated L-type Ca current (I(Ca-L)) and direct activation of epsilonPKC leads to I(Ca-L) inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of epsilonPKC alters I(Ca-L) and its response to adrenergic stimulation. Therefore, we investigated the interaction between beta- and alpha(1)-adrenoceptors vis-à-vis I(Ca-L) in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of epsilonPKC (epsilonPKC agonist). Whole-cell I(Ca-L) was recorded from epsilonPKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) beta-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus alpha(1)-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of epsilonPKC leads to reduced basal I(Ca-L) density. beta-adrenoceptor activation of I(Ca-L) is blunted in epsilonPKC agonist TG mice. alpha-adrenoceptor cross-talk with beta-adrenoceptor signaling pathways vis-à-vis L-type Ca channels is impaired in epsilonPKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in epsilonPKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure.     

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.     

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.     

Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR-1 and PAR-3. In the present study we show that PAR-2 activation by trypsin or by the PAR-2 agonist peptide (SLIGRL) evokes [Ca(2+) ](i) signal in RBCE cells. Taking advantage of RBCE cells expressing PAR-1 and PAR-2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 10(5) -fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood-brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca(2+) ](i) but preincubation of cells with elastase dose-dependently reduced the trypsin-induced [Ca(2+) ](i) signal. Plasmin had a 30% inhibitory effect on the trypsin-induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR-2 is functional in brain capillary endothelium, and that the main fibrinolytic proteases, plasmin and elastase, may regulate PAR-2 signalling under pathological conditions.     

Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-alpha and -beta gene expression

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLalpha and SLbeta mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP(3))/PKC pathways and subsequent activation of the Ca(2+)/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLalpha mRNA expression. Apparently, the downstream Ca(2+)/CaM-dependent cascades were triggered by extracellular Ca(2+) ([Ca(2+)](e)) entry via L-type voltage-sensitive Ca(2+) channels (VSCC) and Ca(2+) release from IP(3)-sensitive intracellular Ca(2+) stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLbeta mRNA expression, except that [Ca(2+)](e) entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLalpha and SLbeta gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.     

Ghrelin suppression of potassium currents in smooth muscle cells of human mesenteric artery

Ghrelin is a 28-amino acid peptide hormone which modulates many physiological functions including cardiovascular homeostasis. Here we report some novel findings about the action of ghrelin on smooth muscle cells (SMC) freshly isolated from human mesenteric arteries. Ghrelin (10(-7) mol/l) significantly suppressed the iberiotoxin-blockable component of potassium currents (I(K)) and depolarized the cell membrane, while having no effect on Ca(2+) currents. Inhibition of inositol-trisphosphate (IP(3))-activated Ca(2+) release channels, depletion of sarcoplasmic reticulum (SR) Ca(2+) stores, blockade of phospholipase D (PLD) or protein kinase C (PKC) each abolished the effect of ghrelin on I(K), while the inhibition of phospholipase C (PLC) did not. These data imply that in human mesenteric artery SMC ghrelin suppresses I(K) via PLD, PKC and SR Ca(2+)-dependent signaling pathway.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.