Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle.

BACKGROUND: Pharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells. METHODS: The effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-ß stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-ß mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter. RESULTS: GKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-ß stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity. CONCLUSIONS: GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity.

Endothelium-dependent vasodilation in myogenically active mouse skeletal muscle arterioles: role of EDH and K(+) channels.

As smooth muscle cell (SMC) membrane potential (E(m)) is critical for vascular responsiveness, and arteriolar SMCs are depolarized at physiological intraluminal pressures, we hypothesized that myogenic tone impacts on dilation mediated by endothelium-derived hyperpolarization (EDH). Studies were performed on cannulated mouse cremaster arterioles [diameter, 33+/-2 microm (n=23) at 60 mmHg; SMC Em -34.6+/-1.2 mV (n=7)]. Myogenic activity was assessed as tone developed in response to intraluminal pressure. Functional observations were related to mRNA, protein expression, and anatomy. Acetylcholine concentration-response curves showed a modest shift following indomethacin (10 microM) and L-NAME (100 microM), although maximal vasodilation was achieved. Residual dilation was removed by apamin (1 microM) in combination with TRAM-34 (1 microM) or charybotoxin (0.1 microM), indicating the requirement of small (S) and intermediate (I) calcium-activated potassium channels (K(Ca)). Charybdotoxin, but not TRAM-34, caused vasoconstriction, presumably through the inhibition of SMC BK(Ca). Expression of SK3 and IK1 was confirmed by immunohistochemistry and polymerase chain reaction, while myoendothelial junctions were common, suggesting a high degree of cell coupling. Also consistent with a role for endothelial K(Ca) channels, acetylcholine increased endothelium [Ca(2 +)](i). Apamin and TRAM-34 similarly blocked EDH-mediated dilation at intraluminal pressures of 30 and 90 mmHg, suggesting that in mouse arterioles, SK(Ca -) and IK(Ca -) mediated mechanisms predominate and operate independently of physiological levels of myogenic activation.

Membrane cholesterol depletion with beta-cyclodextrin impairs pressure-induced contraction and calcium signalling in isolated skeletal muscle arterioles.

OBJECTIVE: Given evidence for clustering of signalling molecules and ion channels in cholesterol-rich membrane domains, the involvement of such structures in arteriolar smooth muscle mechanotransduction was examined. METHOD: To determine the contribution of smooth muscle cholesterol-rich membrane domains to the myogenic response, isolated arterioles were exposed to the cholesterol-depleting agent beta-cyclodextrin (1-10 mM) in the absence and presence of excess exogenous cholesterol. RESULTS: beta-Cyclodextrin significantly impaired pressure-induced vasoconstriction, while excess cholesterol attenuated this effect. Impaired myogenic constriction was evident in de-endothelialized vessels, indicating an action at the level of smooth muscle. beta-Cyclodextrin treatment uncoupled increases in intracellular Ca(2+) from myogenic constriction and depleted intracellular Ca(2+) stores consistent with a loss of connectivity between plasma membrane and sarcoplasmic reticulum signalling. However, beta-cyclodextrin-treated arterioles showed unaltered constrictor responses to KCl and phenylephrine. Electron microscopy verified that beta-cyclodextrin caused a decrease in caveolae, while confirmation of smooth muscle containing caveolae was obtained by immunostaining for caveolin-1. Viability of beta-cyclodextrin-treated arterioles was confirmed by agonist sensitivity and propidium iodide nuclear staining. CONCLUSION: The data suggest that smooth muscle cholesterol-rich membrane domains contribute to the myogenic response. Further studies are required to determine whether this relates to specific mechanosensory events or generalized alterations in membrane function.

Arteriolar myogenic signalling mechanisms: Implications for local vascular function.

Arterioles typically exist in a state of partial constriction that is related to the level of intraluminal pressure. This vasomotor response is a function of the vascular smooth muscle and occurs independently of neurohumoral and endothelial input. The physiological relevance of myogenic constriction relates to the setting of peripheral resistance, provision of a level of tone that vasodilators can access, and a contribution to control of capillary pressure. Despite its importance in the regulation of microvascular haemodynamics the exact cellular mechanisms linking intraluminal pressure to myogenic constriction remain uncertain. Studies using isolated, cannulated arteriole techniques, and freshly dispersed smooth muscle cells, have shown that increased intraluminal pressure/cell stretch leads to smooth muscle cell membrane depolarisation, the opening of L-type voltage-gated Ca2+ channels (VGCC), Ca2+-dependent activation of myosin light chain kinase and actomyosin-based contraction. Questions remain as to how the initial stimulus is detected and how these events lead to membrane depolarisation. A candidate pathway for the mechanosensory events involves the link between extracellular matrix proteins, cell surface integrins and the subsequent activation of intracellular signalling events. Membrane depolarisation may occur through the involvement of various ion channels, including non-selective cation channels (possibly themselves mechanosensitive) that predominantly pass Na+ from the extracellular space. Evidence suggests that this may involve TRP-like channels, possibly TRPM4 or TRPC6 isoforms that are modulated by diacylglycerol and protein kinase C. In addition, the exact roles played by various Ca2+ pools, including those occurring in spatially-restricted domains, and Ca2+ sensitisation, remain uncertain despite the clearly important role of VGCC. Similarly, while a change in intraluminal pressure is associated with the generation of a number of second messengers and the activation of various protein kinases, their roles in myogenic contraction versus long-term adaptive responses, such as tissue remodelling, are still to be defined.

Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.

1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.

Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation.

Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 microM norepinephrine (NE). Prolonged exposure to NE resulted in significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+ buffer, indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 microM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+ dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.