The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT<sub>1B</sub>/5-HT<sub>1D</sub> receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 µ<i>M</i>), and prestimulated with 20 m<i>M</i> K<sup>+</sup> Krebs buffer, 5-HT and CP 93,129, a 5-HT<sub>1B</sub> receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT<sub>1B</sub>/5-HT<sub>1D</sub> receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT<sub>1B</sub> receptor and the 5-HT<sub>1D</sub> receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 ± 3.33 and 52.65 ± 5.34% from basal with 10 µ<i>M</i> 5-HT, respectively). The effect of 10 µ<i>M</i> 5-HT on cAMP was increased in the presence of 20 m<i>M</i> K<sup>+</sup> (reduced by 82.50 ± 2.50 and 87.54 ± 3.97%, respectively). In intact arteries, contraction through 5-HT<sub>1B</sub>/5-HT<sub>1D</sub> receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U01296). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 m<i>M</i> K<sup>+</sup> was present at the onset of stimulation. These data provide the first direct evidence that 5-HT<sub>1B</sub>/5-HT<sub>1B</sub> receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a model system to study further the signalling pathways linked to these receptors.
It has recently been demonstrated that both initiators and inhibitors (viz iloprost) of aggregation stimulate the uptake of [(45)Ca(2+)] by human platelets. Since it was postulated that this calcium uptake reflects changes associated with signal transduction, we investigated the role of cAMP-dependent protein kinases and protein kinase C (PKC) in mediating [(45)Ca(2+)] uptake by washed human platelets. Phorbol myristate acetate (PMA; a PKC activator), sodium fluoride (NaF; a putative G protein activator), ADP and collagen stimulated the uptake of [(45)Ca(2+)] by platelets in dose-dependent manners. The inert phorbol ester, phorbol 4-α-didecanoate had no effect on [(45)Ca(2+)] uptake. PMA-stimulated and NaF-stimulated [(45)Ca(2+)] uptake was inhibited in concentration-dependent manners by the PKC inhibitor, staurosporine. Staurosporine also inhibited [(45)Ca(2+)] uptake when stimulated with collagen, ADP and to a lesser extent by adrenaline. Staurosporine, however, had no effect on [(45)Ca(2+)] uptake when stimulated with calcium ionophore A23187, dibutyryl cAMP or iloprost. The more specific inhibitor of PKC, chelerythrine, inhibited [(45)Ca(2+)] uptake when stimulated by PMA, collagen and adrenaline but not A23187 or dibutyryl cAMP. H8 (a PKA inhibitor) inhibited iloprost- and dibutyryl cAMP-stimulated (but not A23187-stimulated) [(45)Ca(2+)] uptake. These data indicate that [(45)Ca(2+)] uptake is: (1) mediated by PKC when stimulated by proaggregatory agonists and, (2) that cAMP-dependent protein kinase mediated signal transduction involves a calcium uptake component. Thus, these data demonstrate that the [(45)Ca(2+)] uptake elicited by both stimulators and inhibitors of aggregation reflect events associated with signal transduction, possibly at the plasma membrane and not necessarily changes in intracellular calcium (i.e. calcium influx into the cytosol).
Introductionwith thiol groups to produce nitrosothiols [9].Hence, the effects of NO and the pathophysiological influences on it Vascular smooth muscle cell (VSMC) proliferation is an may also be mediated indirectly, potentiated or inhibited important component of vessel wall remodelling in re-through the availability of these oxygen species.Irresponse to injury, for example, after angioplasty or vein spective of the physiological role of NO in VSMC prolifergrafting, and during atherosclerosis formation.Endo-ation, a pharmacological effect of NO may still be thelium-derived nitric oxide (NO) production is both a exploited to reduce neointima formation and prevent tonic and an induced regulator of blood vessel tone [1][2][3].adverse vessel-wall remodeling.We will therefore also Its function is impaired by atherosclerosis and, more consider the experience with this approach either in significantly, by atherogenic risk factors, including hy-conventional or gene therapy.percholesterolaemia, homocysteinaemia, diabetes, smoking and high blood pressure, even before the appearance of overt atherosclerosis [4-6] Endothelial NO production is 2. Alterations in NO formation and NO synthases dysfunctional after balloon injury and in vein grafts at the that may influence VSMC proliferation in time when VSMC proliferation and neointima formation is pathological states progressing [5,6].It has been tempting, therefore, to propose a causal relationship between impaired NO pro-During atherogenesis, VSMCs are activated to a synduction and increased VSMC proliferation.If so, this thetic state, which allows them to proliferate and migrate might explain, in part, the association between endothelial to the intima and produce extracellular matrix [10,11].This dysfunction and atherogenesis.leads to neointima formation and the fibrous component of The primary purpose of this review is to discuss atherosclerosis [12,13].Atherosclerosis is associated with a critically the evidence to support such an hypothesis.We reduced endothelium-dependent vasodilatation, which has will also go on to consider the molecular mechanisms that been ascribed to reduced NO formation [4,14].Although might underlie the inhibitory effects of NO on VSMC endothelium-dependent responses are attenuated in aortic proliferation, with the following important caveats.Firstly, tissue from cholesterol-fed rabbits, total arterial NO proany direct action of NO on an increase in VSMC numbers duction is actually increased [15].This increased NO may be mediated at a variety of levels, for example, on the production is likely to be the result of the induction of signal transduction pathways, on energy production or by inducible nitric oxide synthase (iNOS) in VSMCs [16][17][18][19].promoting cell death.Secondly, in the more complex in Indeed, cholesterol loading of arterial VSMCs upregulates vivo models, effects of NO on endothelial cells (ECs), cytokine-induced NO formation [20].Current evidence platelets and inflammatory cells, rather than directly on indicates that, despite this, the decrease in endothelium-VSMCs may be responsible for modulating VSMC prolif-dependent relaxation is a consequence of increased eration.Thirdly, NO is highly unstable, with a half-life superoxide production, which quenches the increased measured in seconds [2,7,8].It reacts rapidly with oxygen levels of NO [21-23].In a recent elegant study, Luoma et 2 species (O , O and H O ) to produce, nitrite, nitrate or al. [24] found high expression of iNOS in VSMCs from 2 2 2 2the highly reactive species, peroxynitrite (ONOO) and both human and rabbit atherosclerotic lesions, which was co-localised with epitopes of oxidised low-density lipoprotein (LDL) and ONOO-modified proteins.These data
The effect of experimental diabetes mellitus (DM; hyperglycaemic, non‐ketototic; 2 months duration) in the rat on receptor‐linked prostacyclin (PGI 2 ) synthesis (measured as 6‐oxo‐PGF 1α by radioimmunoassay) was studied in the aorta and urinary bladder using adrenaline, angiotensin II (AII) and acetylcholine (ACh). Signal transduction systems were studied via stimulation of PGI 2 synthesis with phorbol ester dibutyrate (PDBU; a protein kinase C activator [PKC]), Ca 2+ ionophore A23187 (A23187) and thapsigargin (both elevate intracellular Ca 2+ , activating phospholipase A 2 [PLA 2 ]) and arachidonate (AA; substrate for PGI 2 synthesis). In response to adrenaline, AII and phorbol ester, aortic PGI 2 release was markedly reduced (all > 75%) in diabetic rats compared to controls. EC 50 s of the dose‐response curves for adrenaline, AII and PDBU were also markedly increased in aortae from DM rats compared to controls. Although there was decreased output of PGI 2 in response to A23187 by aortae from diabetic rats compared to controls, there was no difference in the EC 50 s (mean ± s.e.mean: diabetic, 2.7 ± 0.2 × 10 −6 M; controls 2 ± 0.18 × 10 −6 m ). There were no differences in PGI 2 release (or in the EC 50 s) in response to thapsigargin or AA between aortae from diabetic and control rats. In the urinary bladder, there was a marked increase in PGI 2 output in response to ACh and a marked decrease in EC 50 s for the ACh‐PGI 2 dose‐response curves in diabetic rats (EC 50 = 5.8 ± 0.32 × 10 −7 m ) compared to controls (EC 50 = 2.2 ± 0.15 × 10 −6 m ). Although there was an increase in PGI 2 output in the urinary bladders from diabetic rats in response to A23187, there were no differences in the EC 50 s (control, 1.8 ± 0.2 × 10 −6 m ; diabetic, 1.1 ± 0.15 × 10 −6 m ). In the urinary bladders, there were no differences in PGI 2 output (or the EC 50 s) in response to PDBU, thapsigargin or AA between diabetic or control rats. These data indicate that: (i) reduced PGI 2 synthesis coupled to adrenoceptors and AII receptors in the aortae of diabetic rats may be due to diminished PKC activity and not to changes in receptor density and/or affinity, Ca 2+ stores, PLA 2 , cyclo‐oxygenase or PGI 2 synthase; (ii) the diametrically opposite effect of DM on ACh‐stimulated PGI 2 synthesis is not due to an increase in PKC activity, but possibly to an increase in muscarine receptor number and/or affinity; (iii) changes in receptor‐linked PGI 2 synthesis are not ubiquitous in experimental DM and may be organ‐specific.
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