Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Basic FGF regulates interstitial collagenase gene expression in human smooth muscle cells

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF. bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis. J. Cell. Biochem. 65:32–41. © 1997 Wiley-Liss, Inc.     

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.     

Regulation of CCL5 expression in smooth muscle cells following arterial injury

Chemokines play a crucial role in inflammation and in the pathophysiology of atherosclerosis by recruiting inflammatory immune cells to the endothelium. Chemokine CCL5 has been shown to be involved in atherosclerosis progression. However, little is known about how CCL5 is regulated in vascular smooth muscle cells. In this study we report that CCL5 mRNA expression was induced and peaked in aorta at day 7 and then declined after balloon artery injury, whereas IP-10 and MCP-1 mRNA expression were induced and peaked at day 3 and then rapidly declined.The expression of CCL5 receptors (CCR1, 3 & 5) were also rapidly induced and then declined except CCR5 which expression was still relatively high at day 14 after balloon injury. In rat smooth muscle cells (SMCs), similar as in aorta CCL5 mRNA expression was induced and kept increasing after LPS plus IFN-gamma stimulation, whereas IP-10 mRNA expression was rapidly induced and then declined. Our data further indicate that induction of CCL5 expression in SMCs was mediated by IRF-1 via binding to the IRF-1 response element in CCL5 promoter. Moreover, p38 MAPK was involved in suppression of CCL5 and IP-10 expression in SMCs through common upstream molecule MKK3. The downstream molecule MK2 was required for p38-mediated CCL5 but not IP-10 inhibition. Our findings indicate that CCL5 induction in aorta and SMCs is mediated by IRF-1 while activation of p38 MAPK signaling inhibits CCL5 and IP-10 expression. Methods targeting MK2 expression could be used to selectively regulate CCL5 but not IP-10 expression in SMCs.     

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.     

Force-velocity relationships in hypertensive arterial smooth muscle

Increased total peripheral resistance is the cardinal haemodynamic disorder in essential hypertension. This could be secondary to alterations in the mechanical properties of vascular smooth muscle. Adequate study has not been made of the force-velocity (F-V) relationship in hypertensive arterial smooth muscle. Increased shortening in arterial smooth muscle would result in greater narrowing of arteries. The objectives of this investigation were to see if there is (i) increased shortening or increased maximum change in muscle length (delta Lmax where L stands for muscle length), (ii) an increased maximum velocity of shortening (Vmax) measured in l omicron per second where l omicron is the optimal muscle length for tension development, and (iii) a difference in maximum isometric tension (P omicron) developed in spontaneously hypertensive rat (SHR; N = 6) compared with normotensive Wistar Kyoto rat (WKY;N = 5) caudal artery strips. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed the following: (a) the SHR mean P omicron of 6.21 +/- 1.01 N/cm2 was not different from the mean WKY P omicron of 6.97 +/- 1.64 N/cm2 (p greater than 0.05); (b) the SHR preparations showed greater shortening for all loads imposed; (c) the SHR Vmax of 0.016 l omicron/s was greater than the WKY Vmax of 0.013 l omicron/s (p less than 0.05). This study provides evidence that while hypertensive arterial smooth muscle is not able to produce more force than normotensive arterial smooth muscle, it is capable of faster and greater shortening. The latter could result in increased narrowing of hypertensive arteries and increased blood pressure.     

Phosphorylation of caldesmon in arterial smooth muscle

We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.     

Staurosporine aglycone bilaterally regulates ERK1/2 phosphorylation in rat pulmonary arterial smooth muscle cells

Staurosporine, a protein kinase C (PKC) inhibitor, has been reported to regulate the phosphorylation of ERK1/2 in several cell lines. It is still unknown, however, whether its derivative staurosporine aglycone (SA) has the same effect on ERK1/2 activation. In this study, we investigated the effect of SA on ERK1/2 activity in rat pulmonary arteries and pulmonary arterial smooth muscle cells (PASMCs). The pulmonary arteries and PASMCs were treated with SA at different time points and concentrations, and the activation of ERK1/2 was analyzed by Western blotting. The results showed that SA at nanomolar concentrations suppressed ERK1/2 phosphorylation through the PKC pathway alone, but SA at 30 micromol/L for 2 h enhanced the phosphorylation of ERK1/2. The activation of ERK1/2 was inhibited by the MAPK/ERK kinase inhibitor PD98059 or the protein kinase A (PKA) activator isoproterenol. Together, these results suggest that SA has a strong dual regulating effect on ERK1/2 through the PKC and (or) PKA pathways in rat PASMCs.     

Force regulates smooth muscle actin in cardiac fibroblasts

Chronic ventricular pressure overload can regulate expression of alpha-smooth muscle actin (SMA) in cardiac fibroblasts, but it is unclear if force alone or the concomitant activity of angiotensin II is the principal regulatory factor. To test if SMA mRNA and protein in rat cardiac fibroblasts are regulated directly by force, we first induced SMA expression in cultured cells and then applied magnetically generated perpendicular forces through focal adhesions using collagen-coated magnetite beads. Continuous static forces (0.65 pN/micrometer(2)) selectively reduced SMA but not beta-actin mRNA and protein content within 4 h (to 55 +/- 9% of controls); SMA returned to baseline by 8 h. There was no change in SMA content after force application with either plasma or the cellular fibronectin IIIA domain, BSA, or poly-L-lysine beads. The early loss of SMA was apparently due to selective leakage into the cell culture medium. Treatment with angiotensin II (10 nM) abrogated the force-induced reduction of SMA and increased the levels of this protein. The stress kinase p38 was phosphorylated by force, whereas extracellular signal-regulated kinase 1/2 and c-Jun NH(2)-terminal kinase were unaffected. The p38 kinase inhibitor SB-203580 relieved the force-induced SMA reduction. We conclude that force-induced inhibition of SMA is mediated through the p38 kinase pathway, and this pathway antagonizes angiotensin II regulation of SMA.     

SUMO modification of cell surface Kv2.1 potassium channels regulates the activity of rat hippocampal neurons

Voltage-gated Kv2.1 potassium channels are important in the brain for determining activity-dependent excitability. Small ubiquitin-like modifier proteins (SUMOs) regulate function through reversible, enzyme-mediated conjugation to target lysine(s). Here, sumoylation of Kv2.1 in hippocampal neurons is shown to regulate firing by shifting the half-maximal activation voltage (V(1/2)) of channels up to 35 mV. Native SUMO and Kv2.1 are shown to interact within and outside channel clusters at the neuronal surface. Studies of single, heterologously expressed Kv2.1 channels show that only K470 is sumoylated. The channels have four subunits, but no more than two non-adjacent subunits carry SUMO concurrently. SUMO on one site shifts V(1/2) by 15 mV, whereas sumoylation of two sites produces a full response. Thus, the SUMO pathway regulates neuronal excitability via Kv2.1 in a direct and graded manner.     

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.