BACKGROUND AND PURPOSE Calcitonin gene‐related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin‐1 (ET‐1) from ETA ...receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G‐protein βγ subunits (Gβγ) in the arterial effects of CGRP receptor stimulation.
EXPERIMENTAL APPROACH To test the hypothesis that instead of α subunits of G‐proteins (Gαs), Gβγ mediates the effects of CGRP receptor activation, we used (i) rat isolated mesenteric resistance arteries (MRA), (ii) pharmacological modulators of cyclic nucleotides; and (iii) low molecular weight inhibitors of the functions of Gβγ, gallein and M119. To validate these tools with respect to CGRP receptor function, we performed organ bath studies with rat isolated MRA, radioligand binding on membranes from CHO cells expressing human CGRP receptors and cAMP production assays in rat cultured VSMC.
KEY RESULTS In isolated arteries contracted with K+ or ET‐1, IBMX (PDE inhibitor) increased sodium nitroprusside (SNP)‐ and isoprenaline (ISO)‐ but not CGRP‐induced relaxations. While fluorescein (negative control) was without effects, gallein increased binding of 125I‐CGRP in the absence and presence of GTPγS. Gallein also increased CGRP‐induced cAMP production in VSMC. Despite these stimulating effects, gallein and M119 selectively inhibited the relaxing and anti‐endothelinergic effects of CGRP in isolated arteries while not altering contractile responses to K+ or ET‐1 or relaxing responses to ISO or SNP.
CONCLUSION AND IMPLICATIONS Activated CGRP receptors induce cyclic nucleotide‐independent relaxation of VSMC and terminate arterial effects of ET‐1 via Gβγ.
Real-time polymerase chain reaction (PCR) is commonly used for a sensitive and specific quantification of messenger RNA (mRNA). The levels of mRNA are frequently compared between two or more ...experimental groups. However, such comparisons require normalization procedures, and reference genes are frequently used for this purpose. We discuss pitfalls in normalization and specifically in the choice of reference genes. Reference genes, which prove suitable for some experimental conditions, are not necessarily similarly appropriate for others. Therefore,a proper validation of the suitability of a given reference gene or sets thereof is required for each experimental setting. Several computer programmes are available to aid such validation.
Background and purpose: Recently, some ligands targeting the sphingosine‐1‐phosphate receptor subtype 3 (S1P3) have become available. The characterization of these compounds was mainly based on one ...functional read‐out system, although S1P3 receptors are known to activate different signal transduction pathways. Therefore, this study pharmacologically characterizes these compounds using different assays.
Experimental approach: Using CHO‐FlpIn cells expressing the human S1P3 receptor the potencies and maximal effects of S1P, FTY720‐P, VPC23019, VPC23153 and VPC24191 were determined in three different assays inhibition of cAMP accumulation, elevation of intracellular calcium concentrations (Ca2+i) and S1P3 receptor internalization.
Key results: All compounds tested inhibited forskolin‐induced cAMP accumulation, increased Ca2+i and induced S1P3 receptor internalization but with different potencies and maximal effects. S1P was the most potent compound in all assays followed by FTY720‐P. The VPC compounds were generally less potent than S1P and FTY720‐P. Regarding the maximal effects, all compounds except VPC23153, behaved as full agonists in the cAMP accumulation assay. In the calcium assay, FTY720‐P, VPC23019 and VPC24191 displayed partial and VPC23153 weak partial agonist activity, relative to S1P. Interestingly, treatment with the Gi inactivator Pertussis toxin, did not affect S1P‐induced Ca2+i elevations but inhibited those in response to the other compounds, by about 50%.
Conclusions and implications: This study demonstrated differential response patterns at the S1P3 receptor for a range of ligands. These differences could indicate the presence of functional selectivity at this receptor as FTY720‐P and the VPC compounds seemed to signal predominantly via Gi– whereas S1P activated Gi and Gq‐coupled pathways.
Upon various stimuli, cells metabolize sphingomyelin from the cellular plasma membrane to form sphingosylphosphorylcholine (SPC) or ceramide. The latter can be further metabolized to sphingosine and ...then sphingosine‐1‐phosphate (S1P). Apart from local formation, S1P and SPC are major constituents of blood plasma.
All four sphingomyelin metabolites (SMM) can act upon intracellular targets, and at least S1P and probably also SPC can additionally act upon G‐protein‐coupled receptors. While the molecular identity of the SPC receptors remains unclear, several subtypes of S1P receptors have been cloned and their distribution in cardiovascular tissues is described.
In the heart SMM can alter intracellular Ca2+ release, particularly via the ryanodine receptor, and conductance of various ion channels in the plasma membrane, particularly IK(Ach). While the various SMM differ somewhat in their effects, the above alterations of ion homeostasis result in reduced cardiac function in most cases, and ceramide and/or sphingosine may be the mediators of the negative inotropic effects of tumour necrosis factor.
In the vasculature, SMM mainly act as acute vasoconstrictors in most vessels, but ceramide can be a vasodilator. SMM‐induced vasoconstriction involves mobilization of Ca2+ from intracellular stores, influx of extracellular Ca2+ via L‐type channels and activation of a rho‐kinase.
Extended exposure to SMM, particularly S1P, promotes several stages of the angiogenic process like endothelial cell activation, migration, proliferation, tube formation and vascular maturation.
We propose that SMM are an important class of endogenous modulators of cardiovascular function.
British Journal of Pharmacology (2004) 143, 666–684. doi:10.1038/sj.bjp.0705934
G-protein-coupled receptors constitute one of the largest protein superfamilies in mammals. Since the cloning of the encoding genes, these important drug targets have been subjected to thorough ...biochemical and pharmacological studies. It has become clear that G-protein-coupled receptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important implications for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies.
Increasing evidence suggests a key role for the bioactive lipid sphingosine-1-phosphate (S1P) and its G-protein-coupled receptors (S1P1–5 ) in the cardiovascular system. Recent advances in ...sphingolipid research indicates that cardiomyocyte, vascular smooth muscle and endothelial cell function is greatly influenced by the relative expression and activity both of S1P receptors and of the enzymes involved in sphingolipid metabolism. For instance, the discovery and development of S1P receptor agonists such as FTY720 has clearly indicated the involvement of S1P receptors in the regulation of heart rate. In addition, sphingolipid metabolism induced, for example, by tumour necrosis factor-α or angiotensin II plays an important role in vessel structure, function and tone.
Communication and coordination between primary healthcare professionals and informal caregivers involved in the care for frail older adults is suboptimal and could benefit from interprofessional ...digital communication tools. Implementation in daily practice however frequently fails. We aim to identify generic barriers and facilitators experienced by healthcare professionals and informal caregivers during implementation of interprofessional communication tools to improve their long-term use. Qualitative content analysis using individual semi-structured interviews was used for evaluating three different digital communication tools used by interprofessional primary care networks for frail older adults by 28 professionals and 10 caregivers. After transcription and open coding, categories and themes were identified. Barriers and facilitators were related to: tool characteristics, context of use, involvement of professionals and caregivers. The tool improved availability, approachability and users' involvement. The large number of digital systems professionals simultaneously use, and different work agreements hampered tool use. The tools facilitated care coordination, and professionals declared to be better informed about patients' current situations. Overall, interprofessional digital communication tools can facilitate communication in networks for primary elderly care. However, integration between digital systems is needed to reduce the number of tools. Organizations and policy makers have an important role in realizing the tools' long-term use.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Background and purpose:
The thiazolidine carboxylic acid, BML‐241, has been proposed as a lead compound in development of selective antagonists at the sphingosine‐1‐phosphate receptor (S1P
3
), based ...on its inhibition of the rise in intracellular calcium concentrations (Ca
2+
i
) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML‐241 for the S1P
3
receptor in more detail.
Experimental approach:
Chinese hamster ovary (CHO) cells stably transfected with the S1P
3
, S1P
2
or
α
1A
‐adrenoceptors were used to investigate the effect of BML‐241 on increases in Ca
2+
i
mediated via different receptors. CHO‐K1 cells were used to study ATP‐induced Ca
2+
i
elevations. Effects on S1P
3
‐mediated inhibition of forskolin‐induced cAMP accumulation and on binding to
α
1A
‐adrenoceptors were also investigated. In addition, the effect of BML‐241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath.
Key results:
High concentrations of BML‐241 (10 μM) inhibited the rise in Ca
2+
i
induced by S1P
3
and S1P
2
receptor stimulation; lower concentrations were ineffective. This high concentration of BML‐241 also inhibited Ca
2+
i
increases via P2 (nucleotide) receptor or
α
1A
‐adrenoceptor stimulation. Moreover, BML‐241 (10 μM) inhibited
α
1
‐adrenoceptor‐mediated contraction of rat mesenteric artery but did not displace
3
H‐prazosin from
α
1A
‐adrenoceptors in concentrations up to 100 μM. BML‐241 (10 μM) did not affect the S1P
3
‐mediated decrease of forskolin‐induced cAMP accumulation.
Conclusions and Implications:
We conclude that BML‐241 is a low potency, non‐selective inhibitor of increases in Ca
2+
i
, rather than a specific antagonist at the S1P
3
receptor.
British Journal of Pharmacology
(2006)
149
, 277–282. doi:
10.1038/sj.bjp.0706872
Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, ...we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.
In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA(2), cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (-12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3-5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6-12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).
Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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