dimitrova

K e y w o r d s : grehlin signaling, hormone, orexigenic, prostaglaudin, smooth muscle, tromboxane Furthermore, hormones and local mediators often change the conductivity of ion channels in the cell membrane, which can be used as sensors for proper signaling. Our pilot study showed that ghrelin reduces the iberiotoxin-sensitive Ca 2+ -activated potassium current (I K(Ca) ) elicited in freshly isolated smooth muscle cells of human mesenteric arteries via PLD-and PKCdependent mechanism (15). The sarcoplasmic reticulum is also necessary for this signaling as the blockade of sarco-endoplasmic reticulum Ca 2+ ATPase or IP 3 -activated Ca 2+ channels of internal Ca 2+ stores inhibit the effect of ghrelin on I K(Ca) In the present study, we used pharmacological tools to identify the participants of ghrelin signaling and found a second mediator involved. MATERIALS AND METHODS The investigation conformed to the 'Declaration of Helsinki' 1975Helsinki' (revised 1983. Mesenteric arteries were isolated from extracted specimens of human mesentery taken during abdominal surgery on patients -63 men aged 64.6±1.5 years and 43 women aged 59.3±1.9 -and transported to the laboratory in ice-cold saline. Half of the patients were operated for malignant growths (carcinoma sigma) and the rest -for nonmalignant conditions. Contraction studies Segments of mesenteric arteries were dissected, carefully cleaned of adipose and connective tissues and kept in ice-cold low Ca 2+ solution containing (mmol): 118 NaCl, 5 KCl, 1.2 MgCl 2 , 0.16 CaCl 2 , 10 glucose, 1.2 Na 2 HPO 4 and 24 HEPES. Arterial rings (2 mm long) were mounted on a wire-myograph for isometric tension recording DMT, model 410A (Danish Myo Technology, Aarhus, Denmark) whose chamber was filled with the same ice-cold low Ca 2+ solution. After the mounting of the vessel rings, the organ bath solution was replaced with the same solution containing 2.5 mmol CaCl 2 . The bath was heated up to 37°C and continually bubbled with carbogen (95% O 2 and 5% CO 2 ). The isometric force of contraction was recorded using the program Myodaq (DMT, Aarhus, Denmark). The arterial segments were equilibrated for 1 hour at 37°C in a buffer, which was changed at least 3 times during this equilibration period. In most experiments, the endothelium was removed by careful rubbing with a rat whisker. Then vessels were stretched to their optimal lumen diameter, corresponding to 90% of the passive diameter of the vessel at 100 mm Hg. The viability of the preparations was tested twice by application of 10 µmol noradrenaline. The integrity of the endothelium was tested with 10 µmol acetylcholine added to 10 µmol noradrenaline contracted rings. After the viability tests, the strips were contracted with 1 nmol ET-1, which produced relatively stable isometric contractions allowing the study of the effect of the increasing concentrations of ghrelin. The tension reached a steady state in about 40 minutes after the application of ET-1. Then ghrelin was applied to the bath in increasing concentrations of 10, 30, 100, 300 and 1000 nmol, i.e. starting from a value that is about 10 times higher than its plasma level. It led to a significant effect on native artery preparations (with endothelium and in the absence of TTX) at a 30-100 times higher concentration than in human circulation (11). A possible explanation of this result is that ghrelin has a low diffusion rate through the adventitia, which decreases its interaction with receptors of smooth muscle cells when applied to the bath solution. Other researchers have also used higher ghrelin concentrations while studying the effect of ghrelin on vascular preparations (12), probably due to the same reason. It is also possible that ghrelin reaches such values in the smooth muscle layer of the arterial wall due to its paracrine release from human vascular endothelium (16). Additionally, guinea pigs may have a higher plasma level of ghrelin if compared to humans or rats. The ghrelin-induced changes in tension were expressed as a percentage of the maximum tension elicited by 1 nmol ET-1. The influence of different pharmacological agents (inhibitors) on ghrelin effect was studied by means of their addition to the bath about 40 min after ET-1 application and incubated for about 30 min before the application of ghrelin. The effects of inhibitors were studied using several types in vitro preparations: i) native (untreated) preparations of small human mesenteric arteries; ii) endothelium-denuded preparations; iii) native preparations with tetrodotoxin (TTX, 300 nmol) and mainly iv) endotheliumdenuded and TTX-treated preparations. The time control preparations were equally treated, but instead of ghrelin, an equal volume of solvent (deionised water) was added at the same time intervals. The inhibitors and antagonist were applied to block (to switch off) the studied enzyme or receptor activity and not to induce a partial inhibition only. This forced us to use a higher concentration of these substances. On the other hand, the possibility of a non-specific effect of the pharmacological tools restricted us to using them in lower concentrations. Thus, the aim to choose the optimal concentration of each substance for our experiments was not easy. For almost all of the blockers, however, there are at least several studies on arterial preparations in vitro, in some cases with dose-response curves and/or tests for cross-reaction. Besides, the choice of each concentration was based on our earlier experience with a significant part of the substances used either in electrophysiological or functional studies of different vascular beds. Whole-cell patch-clamp experiments This method has been described in detail elsewhere (17). In brief, whole-cell voltage-clamp experiments were performed on single smooth muscle cells, freshly isolated from human mesenteric arteries. The arteries were cut into 3 mm long pieces and placed in 0.1 mmol Ca 2+ -containing physiological salt solution (PSS, for composition see below) warmed to 37°C and containing 1.5 mg ml -1 collagenase II, 1 mg ml -1 papain, 15 µl ml -1 elastase and 1 mg ml -1 albumin. After 30 to 35 min incubation at 37°C with continuous O 2 bubbling, the enzymes were washed away and the tissue pieces triturated 5 times in Ca 2+ -free PSS using a pipette with a small tip opening. The remainder of the tissue was put back into the enzyme-containing solution for another 5 min and then carefully washed with Ca 2+ -free PSS. Single smooth muscle cells were obtained by gentle trituration in 2 ml of the same Ca 2+ -free solution. Cells could be stored for up to 8 hours in this solution at 4-6°C. The external solution (PSS) for single-cell voltage experiments contained (in mmol): 126 NaCl, 5.6 KCl, 10 HEPES, 20 taurine, 20 glucose, 1.1 MgCl 2 , 0.8 CaCl 2 , 5 Napyruvate and pH was adjusted to 7.4 with NaOH. The same solution was used for the isolation of cells. The solutions in the recording pipette contained (in mmol): 125 KCl, 6 NaCl, 10 HEPES, 1 MgCl 2 , 3 EGTA, 0.1 ATP, 5 Na-pyruvate, 5 succinate, 5 oxalacetate, 5 glucose and 2.15 CaCl 2 to give a calculated free Ca 2+ of 200 nmol and pH was adjusted to 7.4 with KOH. Chemicals Most of the substances used for solution preparation were obtained from ICN (Irvine, CA, USA). NaOH, BSA, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), pertussis toxin, indomethacin, O-(octahydro-4,7-methano-1H-inden-5-yl) carbonopotassium dithioate (D-609), collagenase type II, prostaglandine F 2α (PGF 2α ), (5Z,13E) -(9S,11S,15R)-384 9,15,dihydroxy-11-fluoro-15-(2-indanyl)- Data analysis Current densities were expressed in pA/pF and plotted as functions of the potential applied to obtain data suitable for statistical analysis. The significance of differences between means was assessed using Tukey-Kramer multiple comparison test with p<0.05 regarded significant. The force of contraction was evaluated as a difference in tension (N/m) measured before ET-1 application and the plateau reached afterwards. The contractile effect of ghrelin was expressed as a percentage of the maximal ET-1 induced contraction, taken as 100%. Values were expressed as means±S.E.M. From five to ten human mesenteric arterial preparations (n) were included in the construction of each concentration-response curve of ghrelin. Data were subjected to a comparative statistical analysis one-way ANOVA with Bonferroni correction (p<0.05). RESULTS Octanoyl ghrelin, herein referred to as ghrelin, dosedependently increased the force of contraction of isometric human mesenteric artery preparations constricted with ET-1 The application of increasing doses of ghrelin to external solution containing either NCDC (50 mol) ( The application of increasing concentrations of ghrelin to TTX-and ET-1-containing bath solution failed to influence significantly the force of contraction of endothelium-denuded human mesenteric arteries in the presence of PP2 (10 µmol) -a selective Src family kinase inhibitor The addition of ghrelin (100 nmol) almost entirely inhibited the iberiotoxin-sensitive I K(Ca) recorded during a 500 ms depolarizing pulse to +40 mV from a holding potential of -50 mV (12). Rp-cAMPS (200 µmol), a specific membranepermeable inhibitor of PKA, was without effect on the total outward potassium current (I K ) (n=5), while the subsequent addition of ghrelin (100 nmol) decreased I K to the same degree as in the absence of this PKA inhibitor in single smooth muscle cells from human mesenteric arteries (n=5; p<0.01) DISCUSSION Ghrelin and des-octanoyl ghrelin are equipotent antagonists of ET-1 induced vasoconstriction of human mammary artery (13) while in single smooth muscle cells isolated from human mesenteric arteries des-octanoyl ghrelin blocks the ghrelininduced inhibition of I K(Ca) . This difference supposes the operation of more than one ghrelin receptors in human vascular beds -the des-octanoyl ghrelin-blockable GHS-R1a in human mesenteric arteries and another type in human mammary artery (for a review of ghrelin receptors see 19). Kleinz et al. (13) routinely applied indomethacin to exclude the possibility of endothelium dependent vasodilatation. This treatment however blocks not only endothelial COX1/2 but also those in the tunica media of the artery and thus eliminates the influence of COX1/2 downstream products generated in smooth muscle cells, as suggested by our study. Indeed, such a mechanism is unexpected in blood vessels but is reported in non-vascular smooth muscle (lower esophageal sphincter) that maintains mainly tonic type of contraction similarly to arteries (20). Therefore, it is still difficult to summarize the mechanisms of ghrelin effects on human arteries due to their opposite influences -relaxation and contraction, the different experimental conditions applied and the need for more detailed studies of the intracellular participants. Most often GHS-R1a interacts with heterotrimeric G q/11 proteins and stimulates the G q/11 /PI-PLC/IP 3 +Ca 2+ +DAG/PKC signaling (2). It was reported that the application of a specific PKC inhibitor entirely abolished the effect of ghrelin on I K(Ca) , recorded in single smooth muscle cells of human mesenteric arteries (15). In our study the force of contraction of human mesenteric arteries did not respond to ghrelin application if iberiotoxin or GF109203x were present in the bath solution. Thus, ghrelin requires activation of PKC and suppresses K Ca channels with a large conductance (BK Ca channels) to increase the force of contraction. Ghrelin regulates cAMP/PKA (3-5) and cGMP/PKG signaling (6), which can further influence ion channels. Han et al. (6) reported a ghrelin-induced reduction of voltage-gated I K of rat anterior pituitary tumor (GH3) cells by a PKG-dependent mechanism and Kohno et al. (21) -a ghrelin-induced activation of N-type Ca 2+ channels that required PKA. BK Ca channels are involved in the formation of the spontaneous artery tone, counteract the elevation of the agonist-induced cytosolic free Ca 2+ and participate in the relaxation induced by cAMP/PKAand cGMP/PKG-coupled agonists (22). Therefore, we investigated the participation of both cyclic nucleotides using specific inhibitors Rp-cAMPS for PKA and ODQ for soluble guanylate cyclase. The presence of inhibitors of PKA or soluble guanylate cyclase in the bath solution did not prevent the effect of ghrelin on I K(Ca) . We concluded that the second messengers cAMP and cGMP are not involved in the observed ghrelininduced inhibition of I K(Ca) . On the other hand, in human mesenteric arteries the effect of ghrelin on the force of contraction is blocked by pertussis toxin, which suggests the participation of G i -proteins. In rat islet β-cells ghrelin decreases the insulin secretion by a G αi2 -protein sensitive activation of voltage-gated K + channels and this effect is blocked by D-Lys 3 -GHRP-6, a specific GHS-R1a inhibitor (23). Ghrelin inhibits BK Ca channels in the guinea pig femoral artery via a pertussis toxin and GHS-R1a sensitive pathway (17). Ghrelin also activates G i -protein in cell cultures Ca 2+ released from sarcoplasmic reticulum activates BK Ca channels of smooth muscles cells (24). In human mesenteric arteries IP 3 -sensitive Ca 2+ release is essential for the ghrelininduced decrease of I K(Ca) (15) and for the increase of the force of contraction. Additionally, IP 3 -induced Ca 2+ release from sarcoplasmic reticulum participates in the ET-1 evoked contraction of this vascular bed. Our pharmacological studies suggest that several DAG-producing phospholipases (PI-PLC and PC-PLC) are important in establishing the effect of ghrelin in human mesenteric arteries. We presume that PI-PLC is essential mainly for triggering the contraction (25) and for the IP 3 -induced Ca 2+ release-dependent translocation of PKC to the plasma membrane, while the sustained DAG producer PC-PLC (25) is responsible for the long lasting PKC activation. This suggestion is indirectly supported by the slowly developing inhibition (in 10-14 min) of I K(Ca) by ghrelin in this tissue (15). Using different inhibitors we reveal several new enzymes participating in the ghrelin effect in human mesenteric arteries. Thus, the selective inhibition of MEK or Src kinase entirely blocks the ghrelin-induced contractions in endothelium-denuded human mesenteric arteries with suppressed neurotransmission. The nonselective COX1/2 inhibitor indomethacin eliminates either the ghrelin-induced constriction of human mesenteric arteries or the ghrelin-induced decrease of I K in single smooth muscle cells isolated from the same tissue. All these data suggest the existence of a ghrelin-induced and pertussis toxin-sensitive mechanism, which increases the force of contraction by a consequent activation of Src kinase, MEK and ERK. Similar G iprotein initiated signaling was reported for non-vascular tissues (for review see 26). Next, the stimulated ERK may activate the cytosolic PLA 2 (27), which increases vascular arachidonic acid production -the rate-limiting step for prostaglandin synthesis (28). COX1/2 transform this arachidonic acid into PGE 2 , and then PGE 2 into PGH 2 , which may further yield contracting prostaglandin or thromboxane, as reported for non-vascular smooth muscle It was reported that ghrelin receptor type GHR-R1a has the ability to oligomerize with prostanoid receptors, when they are transiently over-expressed in human embryonic kidney 293 cells (32). The same authors stated that this co-transfection significantly influenced GHR-R1a activity without changes in its affinity for ghrelin. Similarly, as an alternative explanation of our data, it is suggested that ghrelin first binds to GHS-R1a and then activates prostanoid receptor via a direct interaction in the existing GHS-R1a/prostanoid receptor heteromeric complex. If this is the case, the enzymes necessary to demonstrate the effect of ghrelin on the contractile activity (Src kinase, MEK, COX-1 and thromboxane synthase) only support the steady-state thromboxane A 2 production and are not additionally activated by ghrelin. The second explanation of our data, however, seems to be less probable as several new articles report a direct activation of ERK1/2 (3, 7, 33) and Src kinase (34) by ghrelin signaling. Ghrelin decreases the mean arterial pressure of the rat by a COX-insensitive and NOS-sensitive mechanism (35). In rat mesenteric arteries both ghrelin and desacyl ghrelin evoke endothelium-dependent dilation by NOS-and COX-insensitive mechanism (36). Ghrelin inhibits the contraction of human aortic smooth muscle cells by cAMP/PKA pathway activation (4). Ghrelin and desacyl ghrelin antagonize the ET-1-induced contraction of human internal mammary artery (13). Ghrelin decreases the mean arterial pressure in humans as well (11). On the other hand, contractile effects of ghrelin were reported in guinea pig femoral (37) and renal (17) arteries and in rat coronary artery (38). Ghrelin increases the force of contraction of human mesenteric arteries partially constricted with ET-1. The effect is stronger in endothelium-denuded preparations and most pronounced in endothelium-denuded artery segments with blocked action potential propagation of perivascular neurons. These data point to a relaxing effect of ghrelin via endothelium and axonal projections in adventitia, which antagonize the direct and stronger contractile action of ghrelin on the smooth muscle layer of the vascular wall. Additionally, if compared to native preparations during the first half of the experiments, the higher force of contractions of endothelium-denuded human mesenteric arteries suggest that endothelium, as well as perivascular neurotransmission are functional, i.e. they are not badly damaged by the therapy before the surgical intervention or during the transportation. It can be concluded that ghrelin either increases or decreases the force of contraction of arteries depending on their type and the species. Thus, ghrelin and desacyl ghrelin may influence the artery resistance similarly to other regulators of the circulation with opposite effects on different vascular beds. For example, catecholamines redistribute the blood flow throughout the body, depending on the physiological needs, via different adrenergic receptors and intracellular mechanisms. In summary, ghrelin has been shown to increase the force of contraction of human mesenteric arteries by a novel mechanism that requires active Src kinase, MEK, COX-1 and thromboxane synthase and that depends on the release of a local mediator -a T prostanoid receptor agonist. Additionally, our data suggest a novel physiological regulation, in which an empty stomachinitiated increase of ghrelin secretion reduces the abdominal circulation in adult humans until next meal. Acknowledgement

The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava

We characterized the mechanisms in vascular smooth muscle cells (VSMCs) that produce asynchronous, wave-like Ca2+ oscillations in response to phenylephrine (PE). Confocal imaging was used to observe [Ca2+]i in individual VSMCs of intact inferior vena cava (IVC) from rabbits.It was found that the Ca2+ waves were initiated by Ca2+ release from the sarcoplasmic reticulum (SR) via inositol 1,4,5-trisphosphate-sensitive SR Ca2+ release channels (IP3R channels) and that refilling of the SR Ca2+ store through the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) was required for maintained generation of the repetitive Ca2+ waves.Blockade of L-type voltage-gated Ca2+ channels (L-type VGCCs) with nifedipine reduced the frequency of PE-stimulated [Ca2+]i oscillations, while additional blockade of receptor-operated channels/store-operated channels (ROCs/SOCs) with SKF96365 abolished the remaining oscillations. Parallel force measurements showed that nifedipine inhibited PE-induced tonic contraction by 27% while SKF96365 abolished it. This indicates that stimulated Ca2+ entry refills the SR to support the recurrent waves of SR Ca2+ release and that both L-type VGCCs and ROCs/SOCs contribute to this process.Application of the Na+-Ca2+ exchanger (NCX) inhibitors 2′,4′-dichlorobenzamil (forward- and reverse-mode inhibitor) and KB-R7943 (reverse-mode inhibitor) completely abolished the nifedipine-resistant component of [Ca2+]i oscillations and markedly reduced PE-induced tone.Thus, we conclude that each Ca2+ wave depends on initial SR Ca2+ release via IP3R channels followed by SR Ca2+ refilling through SERCA. Na+ entry through ROCs/SOCs facilitates Ca2+ entry through the NCX operating in the reverse mode, which refills the SR and maintains PE-induced [Ca2+]i oscillations. In addition some Ca2+ entry through L-type VGCCs and ROCs/SOCs serves to modulate the frequency of the oscillations and the magnitude of force development

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle.Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing < 10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry.After Ca2+ store depletion with caffeine (10 mm), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 ± 10 and 20 ± 6 s, respectively).To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling.At -80 mV and in 20 mm TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl− current (ICl(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mm; an ICl(Ca) blocker) and to BAPTA AM, but was abolished by 1 μm nifedipine. This nifedipine-sensitive current reversed at +29 ± 2 mV, which shifted to +7 ± 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 μm; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current.At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient ICl(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 μm) during refilling reduced the caffeine-activated ICl(Ca) by 38 ± 5 %. The effect was concentration dependent (1-3000 nm, EC50 64 nm). In Ca2+-free solution, store refilling was similarly depressed (by 46 ± 6 %).Endothelin-1 (10 nm) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 ± 28 nm above basal. Cell Ca2+ was then lowered by 1 μm nifedipine (to 135 ± 22 nm), which reversed on washout.These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine

Parametrization of the contribution of mono- and bidentate ligands on the symmetric C=O stretching frequency of fac-[Re(CO)3]+ complexes

A ligand parameter, IRP(L), is introduced in order to evaluate the effect that different monodentate and bidentate ligands have on the symmetric C≡O stretching frequency of octahedral d6 fac-[Re(CO)3L3] complexes (L = mono- or bidentate ligand). The parameter is empirically derived by assuming that the electronic effect, or contribution, that any given ligand L will add to the fac-[ReCO3]+ core, in terms of the total observed energy of symmetric C≡O stretching frequency (νCOobs), is additive. The IRP(CO) (i.e., the IRP of carbon monoxide) is first defined as one-sixth that of the observed C≡O frequency (νCOobs) of [Re(CO)6]+. All subsequent IRP(L) parameters of fac-[Re(CO)3L3] complexes are derived from IRP(L) = 1/3[νCOobs − 3IRP(CO)]. The symmetric C≡O stretching frequency was selected for analysis by assuming that it alone describes the “average electronic environment” in the IR spectra of the complexes. The IRP(L) values for over 150 ligands are listed, and the validity of the model is tested against other octahedral d6 fac-[M(CO)3L3] complexes (M = Mn, 99Tc, and Ru) and cis-[Re(CO)2L4]+ species and by calculations at the density functional level of theory. The predicted symmetric C≡O stretching frequency (νCOcal) is given by νCOcal = SR[∑IRP(L)] + IR, where SR and IR are constants that depend upon the metal, its oxidation state, and the number of CO ligands in its primary coordination sphere. A linear relationship between IRP values and the well-established ligand electrochemical parameter EL is found. From a purely thermodynamic point of view, it is suggested that ligands with high IRP(L) values should weaken the M−CO bond to a greater extent than ligands with low IRP(L) values. The significance of the results and the limitations of the model are discussed