Mesenteric Vascular Smooth Muscle Cells Research Articles

Agonist-stimulated Ca 2+ transport in mesenteric vascular smooth muscle cells of vitamin D 3-induced calcium overload rats

Altered intracellular Ca 2+ homeostasis and accumulated Ca 2+ deposition in arterial walls contribute to the natural arterial aging and aging-related vascular pathologies. To gain further insight into internal relationship between these two factors, a vitamin D 3-induced vascular Ca 2+ overload rat model was employed. Mesenteric vascular smooth muscle cells (VSMCs) were isolated from both vitamin D 3 and Wistar control rats and were maintained in primary culture for 24 h. Cytosolic and nuclear Ca 2+ ([Ca 2+] i, [Ca 2+] n) in VSMCs were compared between vitamin D 3 and Wistar groups using laser scanning confocal microscopy and Ca 2+-sensitive-dye Fluo-3. Cytosolic and nuclear Ca 2+ were evaluated under both resting and agonist-stimulated conditions including the voltage-dependent Ca 2+ channel openers BayK8644 and KCl, the inositol-1,4,5-trisphosphate (IP 3)-sensitive Ca 2+ release channel activator IP 3, the ryanodine-sensitive Ca 2+ release channel activator trichloromethane, the sarcoplasmic reticulum Ca 2+ -ATPase inhibitor cyclopiazonic acid, and angiotensin II. Although the levels of [Ca 2+] n and [Ca 2+] i were comparable between vitamin D 3 and Wistar groups under the resting condition, the increase of [Ca 2+] n and [Ca 2+] i elicited by various agonists was significantly enhanced in VSMCs from the vitamin D 3 group compared with those from the Wistar group, suggesting abnormality of membrane Ca 2+ gating and intracellular Ca 2+ release under Ca 2+ overload condition. In conclusion, our study indicated that vitamin D 3-induced vascular Ca 2+ overload may directly interrupt cytosolic and nuclear Ca 2+ homeostasis.

Opioid receptor antagonists modulate Ca2+-activated K+ channels in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

Previous study has indicated a significant enhancement of activity of large-conductance Ca2+-activated K+ channel (BKCa) in mesenteric arterial vascular smooth muscle cells isolated from rats in vascular hyporesponsive stage of hemorrhagic shock. In the present study, the effect of opioid receptor antagonism on BKCa activity in the vascular smooth muscle cells of rats in the hyporesponse stage of hemorrhagic shock was investigated by using inside-out configuration of the patch-clamp technique. The results showed that naloxone (10 microM) down-regulated the activity of BKCa by reducing open probability (Po) and open frequency of the channels. The reduction of Po resulted from a decrease of mean open time and an increase of the slow closed time constant. Naltrindole and nor-binaltorphimine (100 nM) had the similar effects to that of naloxone, but no significant effect of beta-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that delta- and kappa-opioid receptors, but not mu-receptors, may be involved in the regulation of BKCa in vascular hyporesponse stage, and that inhibition of BKCa may be one of the mechanisms of the opioid receptor antagonists improving the response of resistance arteries to vasoactive stimulants during the decompensatory stage of hemorrhagic shock.

Upregulation of L-type Ca2+ channels in mesenteric and skeletal arteries of SHR.

An increased Ca2+ influx attributed to dihydropyridine-sensitive L-type Ca2+ channels has been demonstrated in mesenteric vascular smooth muscle cells of spontaneously hypertensive rats (SHR). This study examined whether an upregulation of the pore-forming alpha1C subunit of the L-type Ca2+ channel underlies this ionic defect. With the use of mesenteric arcade arteries from 12- to 16-week-old SHR and normotensive Wistar Kyoto (WKY) rats, reverse transcriptase-polymerase chain reaction demonstrated an increased level of amplified cDNA corresponding to the alpha1C subunit mRNA in the SHR arteries. Western blots confirmed that the increased mRNA expression was associated with a 3.4-fold increase in the immunoreactive signal of the alpha1C subunit protein in SHR compared with WKY mesenteric arteries, and immunocytochemistry confirmed this abnormality at the single-cell level. Finally, isolated mesenteric arteries from SHR were highly reactive to Bay K8644 and developed anomalous Ca2+-dependent tone, suggesting a functional role for alpha1C subunit upregulation in vascular hyperreactivity. To determine if these Ca2+ channel abnormalities extended to the SHR skeletal muscle bed, we repeated a similar series of studies in WKY and SHR hind limb arteries. Skeletal muscle arteries from SHR also expressed higher levels of alpha1C subunit mRNA and protein than WKY arteries and developed anomalous Ca2+-dependent tone attributed to L-type Ca2+ channels. Our data provide the first evidence that the alpha1C subunit mRNA and protein are upregulated in SHR arteries and that the increased numbers of L-type Ca2+ channel pores are associated with the generation of abnormal vascular tone.

Increased expression of peroxisome proliferator-activated receptor-alpha and -gamma in blood vessels of spontaneously hypertensive rats.

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors that include PPAR-alpha, PPAR-gamma, and PPAR-delta. We hypothesized that PPAR expression in blood vessels could be reduced in hypertension to result in increased vascular growth and reduced apoptosis. We investigated the abundance of PPAR-alpha and PPAR-gamma in aorta and mesenteric arteries from young (6-week-old) and adult (16-week-old) spontaneously hypertensive rats (SHR) compared with age-matched control Wistar-Kyoto rats (WKY). mRNA levels of PPAR-alpha and PPAR-gamma were determined by reverse transcription-polymerase chain reaction. Protein expression was evaluated by Western blot and by immunohistochemistry. PPAR-gamma was expressed in aortic and mesenteric vascular smooth muscle cells (VSMCs) from intact tissue and cultured cells. PPAR-alpha was expressed in intact vascular tissue but was almost undetectable in cultured VSMCs. In mesenteric arteries from adult SHR, PPAR-alpha and PPAR-gamma mRNA levels were significantly greater than in WKY (P<0.05). In aorta, PPAR-alpha mRNA was significantly (P<0.05) more abundant in adult (but not in young) SHR than in WKY, whereas there was no difference in PPAR-gamma mRNA between WKY and SHR. PPAR-alpha and PPAR-gamma mRNA were greater in mesenteric arteries (P<0.05) in young and adult SHR than in WKY. Expression of PPAR-alpha and PPAR-gamma was similar in SHR and WKY in other tissues. In cultured mesenteric VSMCs, PPAR-gamma mRNA was 3-fold higher in SHR than in WKY. Immunohistochemistry demonstrated that PPAR-gamma resided constitutively in the cytoplasm in primary and low-passaged aortic and mesenteric VSMCs, whereas PPAR-alpha was almost undetectable. Thus, aorta and mesenteric resistance arteries from SHR in the prehypertensive and the established phase of hypertension exhibit increased expression of both PPAR isoforms, whereas other tissues do not. Changes (increases) in PPAR expression may play a compensatory role in the remodeling of blood vessels in SHR.

Cross talk between prostacyclin and nitric oxide under shear in smooth muscle cell: role in monocyte adhesion.

We tested the hypothesis that at sites of vascular damage, vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO) in vascular smooth muscle cells (VSMC). Confluent A7r5 cells derived from rat aortic VSMC and mesenteric VSMC were exposed to shear stress at 15 dyn/cm(2) for 90 min with the use of a cone-plate device, and productions of prostacyclin and NO were examined. Shear stress increased cumulative production of prostacyclin by 3- to 3.5-fold and that of NO by 6- to 7.5-fold. Western blot analysis showed that inducible NO synthase protein was expressed after shear stress in both types of VSMC. Inhibition of NO synthase enhanced the shear-induced production of prostacyclin from 40 to 60%. Shear-induced production of NO was suppressed by 70% after treatment with 10(-4) M of indomethacin. A7r5 cells adhesiveness for monocytes was suppressed by 50% after shear stress. This suppression was abolished by pretreatment with 10(-4) M of indomethacin, whereas inhibition of NO synthase only minimally inhibited it. We conclude that there is a cross talk of shear-induced production of prostacyclin and NO in VSMC. At sites of vascular damage, prostacyclin synthesis may prevent monocyte adhesiveness for VSMC through the concomitant enhancement of NO production.

Docosahexaenoic acid, a peroxisome proliferator-activated receptor-alpha ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase.

Omega-3 fatty acids (n-3 FAs) have been shown to exert a blood pressure-lowering effect in hypertension, possibly in part by influencing vascular structure. We previously demonstrated that n-3 FAs induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on the structure of blood vessels. In the present study, we investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured mesenteric VSMCs from Sprague-Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FAs. Morphological changes in apoptosis and DNA fragmentation were examined with phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, we evaluated the expression of phosphorylated p38 mitogen-activated protein kinases, bax, bcl-2, cytochrome c, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) with Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing, and apoptotic bodies in VSMCs. DHA time-dependently activated p38 mitogen-activated protein kinases, bax, PPAR-alpha, and cytochrome c, with maximal effects obtained after 5 and 30 minutes and 1 and 3 hours, respectively. SB-203580 and SB-202190, selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPAR-alpha but had no effect on the expression of bax or cytochrome c. The present results indicate that DHA induces apoptosis in VSMCs through >/=2 distinct mechanisms: (1) a p38-dependent pathway that regulates PPAR-alpha and (2) a p38-independent pathway via dissipation of mitochondrial membrane potential and cytochrome c release. The death-signaling pathway stimulated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA may play a pathophysiological role in vascular remodeling in cardiovascular disease.

P38 MAP Kinase Regulates Vascular Smooth Muscle Cell (VSMC) Collagen Synthesis by Ang II in Shr But not in Wky.

P81 Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Molecular mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38, may be important. We studied the role of ERK1/2 and p38 in vascular growth effects mediated by Ang II in SHR. Cultured mesenteric VSMC from WKY and SHR were used. Phosphorylation of ERK1/2 and p38 was assessed by Western blot using phospho-specific antibodies. c-fos mRNA expression was determined by RT-PCR. Ang II-stimulated DNA, protein and proline synthesis were assessed as indices of VSMC proliferation, hypertrophy and collagen production and were determined by measuring incorporation of 3 H-thymidine, 3 H-leucine and 3 H-proline respectively. Ang II dose-and time-dependently increased ERK1/2 and p38 phosphorylation. Responses were increased (p c-fos mRNA expression in SHR (173 ±12 % of control). These actions were inhibited by PD98059, but not by SB202190. Ang II stimulated 3 H-thymidine, 3 H-leucine and 3 H-proline incorporation. Responses were enhanced (p 1 receptor antagonist, but not by PD123319, AT 2 receptor blocker. Thus 1)Ang II-mediated activation of vascular ERK1/2 and p38 is increased in SHR, 2) augmented c-fos and growth responses are generated primarily via ERK1/2, 3) p38 influences Ang II-induced collagen production in SHR but not in WKY. In conclusion, these results indicate differential roles of ERK1/2 and p38 in AT 1 -stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in genetic hypertension. (Supported by a research grant from Bristol-Myers Squibb/Sanofi)

Docosahexaenoic Acid, a Peroxisome Proliferator-Activated Receptor-α Activator, Induces Apoptosis in Vascular Smooth Muscle Cells by Activation of P38 Mitogen-Activated Protein Kinase

9 Omega-3 fatty acids (n-3 FAs) exert a blood pressure-lowering effect in hypertension, possibly by influencing vascular structure. We previously demonstrated that n-3 FAs might induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on structure of blood vessels. This study investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured Mesenteric VSMCs from Sprague Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FA. Morphological changes of apoptosis and DNA fragmentation were examined by phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, expression of phosphorylated p38 mitogen-activated protein kinases (p38 MAPKs), bax, bcl-2, cytochrome C and peroxisome proliferator-activated receptors-α (PPARs-α) was evaluated by Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing and apoptotic bodies in VSMCs. DHA increased apoptosis (%) in a time-dependent manner to 1.5±0.1, 3.6±0.5, 7.1±0.4, 22.5±0.6, 50.8±1.8 and 61.4±0.9 after 0, 1, 3, 6, 17, and 24 h, respectively. DHA time-dependently activated p38 MAPKs, bax, PPARs-α and cytochrome C with maximal effects obtained after 5, 30 min, 1 h and 3 h, respectively to 551±42, 245±55, 310±12 and 407±14.7 % of controls, respectively. SB-203580 (10 -5 M) and SB-202190 (10 -5 M), selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPARs-α, but had no effect on expression of bax or cytochrome C. The present results indicate that DHA induces apoptosis in VSMCs through at least two distinct mechanisms: (i) a p38-dependent pathway that regulates PPAR-α and (ii) a p38-independent pathway via dissipation of mitochondrial transmembrane potential. The death-signaling pathway mediated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA could play a pathophysiological role in vascular remodeling in cardiovascular disease.

P38 MAP Kinase Regulates Vascular Smooth Muscle Cell (VSMC) Collagen Synthesis by Ang II in Shr But not in Wky.

P81 Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Molecular mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38, may be important. We studied the role of ERK1/2 and p38 in vascular growth effects mediated by Ang II in SHR. Cultured mesenteric VSMC from WKY and SHR were used. Phosphorylation of ERK1/2 and p38 was assessed by Western blot using phospho-specific antibodies. c-fos mRNA expression was determined by RT-PCR. Ang II-stimulated DNA, protein and proline synthesis were assessed as indices of VSMC proliferation, hypertrophy and collagen production and were determined by measuring incorporation of 3 H-thymidine, 3 H-leucine and 3 H-proline respectively. Ang II dose-and time-dependently increased ERK1/2 and p38 phosphorylation. Responses were increased (p&lt;0.01) 3-4 fold in SHR. PD98059, selective MEK1/2 inhibitor, and SB202190, selective p38 inhibitor, abolished Ang II-induced ERK1/2 and p38 activation respectively. Ang II increased (p&lt;0.05) c-fos mRNA expression in SHR (173 ±12 % of control). These actions were inhibited by PD98059, but not by SB202190. Ang II stimulated 3 H-thymidine, 3 H-leucine and 3 H-proline incorporation. Responses were enhanced (p&lt;0.02) 2-3 fold in SHR. PD98059 attenuated Ang II-induced growth effects and normalized responses in SHR. SB212190 did not alter Ang II-elicited DNA synthesis, but reduced (P&lt;0.05) collagen production in SHR. Effects were inhibited by irbesartan, AT 1 receptor antagonist, but not by PD123319, AT 2 receptor blocker. Thus 1)Ang II-mediated activation of vascular ERK1/2 and p38 is increased in SHR, 2) augmented c-fos and growth responses are generated primarily via ERK1/2, 3) p38 influences Ang II-induced collagen production in SHR but not in WKY. In conclusion, these results indicate differential roles of ERK1/2 and p38 in AT 1 -stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in genetic hypertension. (Supported by a research grant from Bristol-Myers Squibb/Sanofi)

Antiproliferative effect of L-NAME on rat vascular smooth muscle cells

The nitric oxide synthase (NOS) inhibitor L-NAME may have growth inhibitory effects in vivo. We investigated in vitro the potential growth inhibitory effects of three different NOS inhibitors: L-NAME (1 mM), LNMMA (1 mM) and aminoguanidine (0.5 mM), on fetal bovine serum (FBS) and platelet derived growth factor (PDGF-BB)-stimulated growth in cultured vascular smooth muscle cells (VSMCs). [ 3H]-thymidine incorporation into rat mesenteric VSMCs was measured as an index of VSMCs proliferation (DNA synthesis) and activation of extracellular signal regulated kinase (ERK1/2), a major signaling event in cell growth, was measured by western blot assay. PDGF-BB (0–5 ng/mL) and FBS (0–5%) increased [ 3H]-thymidine incorporation in a dose-dependent manner up to 6–10 fold. L-NAME significantly reduced PDGF-BB (5 ng/ml) and FBS (5%) stimulated DNA synthesis by 46% and 38% respectively. The increase of [ 3H]-thymidine incorporation induced by PDGF-BB and FBS was unaltered by L-NMMA. In contrast, aminoguanidine induced an increase in FBS and PDGF-BB-stimulated [ 3H]-thymidine incorporation of 64% and 34% respectively above cells not exposed to aminoguanidine. ERK1/2 phosphorylation induced by PDGF-BB and FBS was not affected by pre-treatment with L-NAME or aminoguanidine. In conclusion, NOS inhibitors differentially influence DNA synthesis in VSMCs: L-NAME inhibits FBS and PDGF-BB-stimulated cellular proliferation whereas aminoguanidine accentuates FBS and PDGF-BB-stimulated VSMCs proliferation. These phenomena are independent of the ERK1/2 pathway. The growth inhibitory effects of L-NAME may be related to differences in properties from other NOS inhibitors, and independent of its ability to inhibit NOS.

Endothelin-1 attenuates omega3 fatty acid-induced apoptosis by inhibition of caspase 3.

Endothelin-1 (ET-1) may be involved in the induction of vascular hypertrophy in hypertension. ET-1 may also modulate vascular growth through the exertion of antiapoptotic effects. The omega3 fatty acids (omega3 FAs), which have antiproliferative effects in various cell types, may have a beneficial role in hypertension. We tested the hypothesis that ET-1 could act as a survival factor against omega3 FA-induced apoptosis and attempted to elucidate possible molecular mechanisms underlying the protective action of ET-1 on docosahexaenoic acid (DHA)-induced apoptosis. Mesenteric vascular smooth muscle cells were stimulated with DHA, a representative omega3 FA. Dose-response curves of DHA at different apoptotic stages were assessed with the use of flow cytometry: (1) very early: plasma membrane phosphatidylserine (PS) translocation; (2) early: change in mitochondrial transmembrane potential (DeltaPsim); and (3) late: cell cycle analysis. Expression of the proapoptotic protein bax and the antiapoptotic protein bcl-2 was determined with Western blot assay. The activity and the expression of caspase 3, which is a critical proteolytic enzyme involved in the death-signaling pathway, were evaluated with a fluorometric immunosorbent enzyme assay and Western blot analysis, respectively. Apoptosis, which was detected with PS translocation, DeltaPsim disruption, and cell cycle analysis, was increased dose dependently by DHA. DHA-induced apoptosis was attenuated through exposure to ET-1 for 1 hour before DHA in cell cycle analysis. The interference of ET-1 with DHA-induced apoptosis, as detected with cell cycle analysis, was not apparent at the membrane (PS translocation) or the mitochondrial (DeltaPsim) level. The increase in bax/bcl-2 ratio in DHA-stimulated cells was not affected by ET-1. However, DHA increased both caspase 3 activity and the active forms of caspase 3 (20 and 17 kDa), resulting in enhanced DNA fragmentation as shown through Hoechst staining and fluorescence microscopy, which were attenuated by ET-1 pretreatment. In conclusion, DHA, an omega3 FA, induced apoptosis in vascular smooth muscle cells in a dose-dependent manner. ET-1 exerted important protective effects through the attenuation of DHA-induced caspase 3 activation and subsequent DNA fragmentation in the late stages of apoptosis.

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.

Proteoglycan production by vascular smooth muscle cells from resistance arteries of hypertensive rats.

Extracellular matrix (ECM) modifications in the vascular wall contribute to the narrowing of arteries in hypertension. Because direct evidence for the role of proteoglycans (PGs) in the pathological process of resistance-sized arteries has not already been demonstrated, we examined the effect of growth factors on secreted and membrane-bound PG synthesis by cultured mesenteric vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and Wistar rats. After 48 hours of stimulation with angiotensin II (Ang II), platelet-derived growth factor (PDGF-BB), and 10% fetal calf serum (FCS) or 0.1% FCS as control, PG synthesis (in dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method with both [(3)H]-glucosamine and [(35)S]-sodium sulfate, which are incorporated into all complex carbohydrates or only into sulfated disaccharides, respectively. VSMC from SHR displayed a significantly lower level of synthesis of M-ECM [(3)H]-PGs than those of Wistar rats in all the experimental groups, including the control group (0. 1% FCS), but no differences in M-ECM [(35)S] uptake were found in any case. In the P-ECM, Ang II was the only factor that produced a lesser effect on [(3)H]-glucosamine and a greater effect on [(35)S]-sodium sulfate uptakes in VSMC from SHR than from Wistar rats. The most prominent change seen in VSMC from SHR was an increased sulfation, assessed by [(35)S]/[(3)H] ratio, in nonstimulated cells and in response to 10% FCS and Ang II but not to PDGF-BB compared with VSMC from Wistar rats. These data indicate the existence of changes in PG modulation in the resistance vessels of SHR, which suggests that PGs may contribute to the development of structural and functional modifications in hypertensive states.

Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells.

1. The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT-PCR, cDNA cloning and expression techniques. 2. RT-PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found. 3. Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n = 3; P < 0.05). A two-fold difference was detected between Kir2.1 mRNA and beta-actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations. 4. Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+. 5. The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half-block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes. 6. Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle.

The direct effects of thyroid hormones on rat mesenteric resistance arteries.

The direct relaxant effects of thyroid hormones on mesenteric resistance vessels were investigated using an isometric wire myograph. Both the L- and the D-isomers of thyroxine (T4) and triiodothyronine (T3) were studied. In contrast with the long-term effects of thyroid hormones, both T4 enantiomers proved more potent in inducing vascular relaxation than the two T3 enantiomers. The interaction between thyroid hormones and calcium-induced contractions was studied. T4 concentration dependently inhibited the Ca2+ induced contractions, showing noncompetitive interaction. Furthermore, we investigated whether the endothelium was involved in the relaxant effect to L-T4. The T4 induced relaxation proved impaired by prior incubation with the nitric oxide (NO) inhibitor N-omega-nitro-L-arginine methylester HCl (L-NAME, 0.1 microM), indicating that T4 is able to stimulate the production of endothelium-derived NO. L-T4-induced relaxation was enhanced by prior incubation with indomethacin (10 microM), whereas in endothelium-denuded preparations an unaltered response was found. The present results indicate that L-T4-induced relaxation is established by an indirect effect via the endothelium and by a direct effect on vascular smooth muscle cells, possibly by influencing calcium fluxes. Because vascular relaxation is established at supraphysiologic concentrations (approximately 100 times the basal level) of thyroid hormone, it is concluded that the direct effect of thyroid hormone on mesenteric vascular smooth muscle cells are not relevant for the in vivo situation.

Regulation of endothelin receptors by nitric oxide in cultured rat vascular smooth muscle cells

Two important mediators of endothelium-dependent regulation of vascular smooth muscle tone and proliferation are nitric oxide (NO) and endothelin (ET-1). An imbalance between NO and ET-1 may contribute to the alterations in vascular tone characteristic of cardiovascular disease. The objective of this study was to determine whether NO regulates ET receptors in cultured rat superior mesenteric artery vascular smooth muscle cells (RVSMC). Chronic treatment of quiescent RVSMC with any one of three chemically dissimilar NO-generating drugs, S-nitroso-N-acetyl penicillamine (SNAP), sodium nitroprusside (SNP), and isosorbide dinitrate (ISDN) produced a significant dose- and time-dependent increase in the number of ET-A receptors, while concomitantly increasing the affinity of ET-1 for this receptor. This effect was mimicked by both 8-bromo-cGMP and 8-bromo-cAMP. The requirement of both protein and RNA synthesis and activation of a cAMP-dependent protein kinase (A-kinase) was demonstrated following inhibition of this regulation by cycloheximide, actinomycin D and KT5720 (a specific A-kinase inhibitor), respectively. In addition, the cytokine interleukin 1 beta (IL-1 beta) which induced NOS activity with subsequent NO synthesis in vascular smooth muscle, also caused a similar upregulation of ET receptors. This effect was attenuated in the presence of the specific NOS inhibitor, L-NAME. To assess the possible functional consequences of this NO-mediated upregulation, the effect of SNAP pretreatment on isolated vessel reactivity was determined. In both superior mesenteric artery and thoracic aorta rings, SNAP pretreatment caused a significant increase in the maximal force of contraction to ET-1. Collectively, these data suggest that NO regulates ET-A receptors in vitro through a cGMP-dependent mechanism via activation of the cAMP-dependent protein kinase. We conclude that a similar interaction between NO and ET-1 may be operational in vivo.

ETA AND ETB RECEPTORS ON VASCULAR SMOOTH MUSCLE CELLS FROM MESENTERIC VESSELS OF SPONTANEOUSLY HYPERTENSIVE RATS

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.

Mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells

To determine whether changes in haemodynamic load, simulated in vitro by mechanically stretching cultured vascular smooth muscle cells, could be transduced into biochemical signals similar to those produced by growth factors. A system was developed which was capable of stretching cultured vascular smooth muscle cells from 0 to 20%. The effect of stretching quiescent vascular smooth muscle cells on both c-fos messenger RNA (mRNA) expression and release of total inositol phosphates was determined over a time interval of 0-360 min. Rat mesenteric artery vascular smooth muscle cells were grown using standard cell culture methods. Induction of the proto-oncogene, c-fos, was determined by Northern blotting. Phosphoinositide breakdown was assessed by measuring [3H]-inositol phosphates released from prelabelled cells. A 20% fixed stretch resulted in a rapid induction of c-fos mRNA which reached maximal levels by 15 min. The amount of c-fos mRNA detected was dependent on the degree of stretch, with maximum induction obtained for 15 and 20% stretch. The effects of mechanical stretch were also assessed on phosphoinositide turnover by measuring [3H]-inositol phosphates released from prelabelled cells. A 20% fixed stretch of vascular smooth muscle cells for 20 min resulted in a 3.2-fold increase in total [3H]-inositol phosphates released compared with unstretched cells. Our results show that mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells in vitro. These observations suggest that mechanical stretch and growth factors share common signal transduction pathways which may be important in the development of vascular hypertrophy.

Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1

To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats. Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology. Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100 s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups. In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Ang II are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.

Phenoxybenzamine mediated inhibition of the vascular dopamine D 1 receptor

Cultures of rat mesenteric artery vascular smooth muscle cells express both vascular dopamine D 1 receptors and β 2-adrenoceptors but not α 1- or α 2-adrenoceptors, permitting direct investigation of the dopamine D 1 receptor antagonist activity of phenoxybenzamine. After incubating cells with phenoxybenzamine (10 −5 M) for 20 min, an 80% inhibition of dopamine-induced (10 −4 M) cAMP formation was observed. Isoprenaline-induced (10 −5 M) cAMP formation remained unaffected by phenoxybenzamine. Inhibition of the dopamine response following 20 min incubation with phenoxybenzamine, was concentration-related and could not be reversed by repeated washing. Mean IC 50 (95% confidence limits) = 4.68 x 10−6 M (3.86−5.01). Exposure of cells to the selective dopamine D 1 receptor partial agonist (+)-SKF 38393 (10 −6 M) prior to phenoxybenzamine incubation, resulted in protection of dopamine-induced cAMP formation. Exposure of cells of the stereo-enantiomer (−)-SKF 38393 (10 −6 M) did not produce any protective effect. The concentration-effect curve for (+)-SKF 38393 mediated protection had a mean EC 50 value of 0.11 × 10 −6 M (0.10−0.11), which is comparable with the K a apparent value (0.06 × 10 −6 M) for this compound when acting as an agonist to induce cAMP formation via the vascular dopamine D 1 receptor. Previous studies of the vascular dopamine D 1 receptor are likely to have been influenced by the frequent use of phenoxybenzamine, which we have shown to act as a potent antagonist at this site.