Biomedical subjects

Timothy V Murphy

Publications and source records attributed to Timothy V Murphy.

7 recordsLinked to original sources

PubMed2006

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.

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PubMed2003

Intraluminal pressure stimulates MAPK phosphorylation in arterioles: temporal dissociation from myogenic contractile response.

Members of the MAPK family of enzymes, p42/44 and p38, have been implicated in both the regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response after increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Experiments used single cannulated arterioles isolated from the cremaster muscle of rats with some vessels loaded with the fluorescent Ca2+-sensitive dye fura 2 (2 microM). The p42/44 inhibitor PD-98059 (50 microM) caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca2+. Western blotting revealed a significant increase in the level of phosphorylation of p42/44 15 min after the application of a 30- to 100-mmHg pressure step. Phosphorylation of p42/44 was a late event that appeared to be temporally dissociated from contraction, which was complete within 1-5 min. EGF (80 nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor SB-203580 (10 microM) did not alter baseline diameter, nor did it prevent myogenic vasoconstriction. Consistent with these observations, SB-203580 did not cause a measurable change in intracellular Ca2+. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent the activation of compensatory, but parallel, pathways, including those related to growth and remodeling.

Adenosine↗

PubMed2003

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.

Animals↗

PubMed2002

Mechanisms underlying pervanadate-induced contraction of rat cremaster muscle arterioles.

The current study examined the role of extracellular Ca2+, calmodulin and myosin light-chain kinase (MLCK) in pervanadate-induced constriction of cannulated, pressurized rat cremaster arterioles. Pervanadate (0.03-100 microM) induced a concentration-dependent constriction of arterioles that was significantly attenuated (P<0.05) by the tyrosine kinase inhibitor tyrphostin 47 (30 microM). The L-type voltage-sensitive Ca2+ channel antagonists verapamil (10 microM) and nifedipine (1 microM) dilated vessels possessing myogenic tone but had no demonstrable effect on pervanadate constriction, while a higher concentration of nifedipine (10 microM) reduced constriction by approximately 50%. Pervanadate-induced contractions were reduced by the calmodulin inhibitor W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide, 50 microM) and the MLCK inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine, 10 or 30 microM). Removal of extracellular Ca2+ abolished the contractile effect of pervanadate. Measurement of changes in arteriolar wall [Ca2+] using the Ca2+ sensitive dye Fura-2 showed that pervanadate did not increase [Ca2+] during arteriolar constriction. These observations suggest that pervanadate-induced contraction of smooth muscle in the cremaster arteriole involves Ca2+/calmodulin-dependent myosin phosphorylation and possibly sensitization of the contractile apparatus to Ca2+.

Animals↗

PubMed2002

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.

Adenosine Triphosphate↗

PubMed2002

Capacitative Ca(2+) entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C.

1. Agonists increase endothelial cell intracellular Ca(2+), in part, by capacitative entry, which is triggered by the filling state of intracellular Ca(2+) stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca(2+) stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP(3) receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP(3) receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca(2+) entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3. 2APB (30 - 300 microM) inhibited Ca(2+) entry induced by both agonists (ATP 1 microM, bradykinin 0.1 microM) and receptor-independent mechanisms (thapsigargin 1 microM, ionomycin 0.5 and 5 microM). 2APB did not diminish endothelial cell ATP-induced production of IP(3) nor effect in vitro binding of [(3)H]-IP(3) to an adrenal cortex binding protein. Capacitative Ca(2+) entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca(2+) release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3 - 10 microM) inhibited ATP-induced Ca(2+) release and capacitative Ca(2+) entry. Further, xestospongin C inhibited capacitative Ca(2+) entry induced by thapsigargin (1 microM) and ionomycin (0.5 microM). 5. The data are consistent with a mechanism of capacitative Ca(2+) entry in vascular endothelial cells which requires (a) IP(3) receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca(2+) release and entry pathways.

Actins↗

PubMed2002

Cellular signalling in arteriolar myogenic constriction: involvement of tyrosine phosphorylation pathways.

1. An increase in transmural pressure in arterioles results in a shortening of vascular smooth muscle cells, with subsequent constriction of the vessel. The mechanisms underlying this myogenic contraction are not fully understood; however, the obligatory role of increases in intracellular [Ca(2+)] and myosin light chain phosphorylation have been demonstrated. 2. The myogenic response shows a relationship with smooth muscle cell membrane potential and influx of extracellular Ca(2+) through voltage-operated Ca(2+) channels (VOCC). Mechanically sensitive channels and possibly release of Ca(2+) from intracellular stores may play a role. However, there are other components of myogenic contraction that cannot be explained by a Ca(2+)-MLCK mechanism, for example the initial sensing of alterations in transmural pressure, whether sustained myogenic constriction involves myofilament Ca(2+) sensitization or remodelling of the vessel wall in response to a maintained increase in transmural pressure. 3. In an attempt to investigate these areas, recent studies have examined a role for tyrosine phosphorylation pathways in pressure-induced contraction of arterioles. In rat pressurized cremaster arterioles, tyrosine kinase inhibitors dilated vessels showing spontaneous myogenic tone and tyrosine phosphatase inhibitors caused vasoconstriction. However, pressure-induced myogenic constriction of vessels persisted in the presence of these agents. Biochemical studies revealed that phosphotyrosine formed at a relatively slow rate (significant after 5 min, with maximal increase after approximately 15 min) in response to increased vessel transmural pressure, in contrast with myosin light chain phosphorylation or the time-course of myogenic constriction itself (maximum within 1 min). 4. Taken together, these observations support the idea of a role for tyrosine phosphorylation pathways in longer-term responses to increased transmural pressure rather than acute myogenic constriction. Phosphotyrosine formation was also more closely correlated to vessel wall tension (pressure x diameter) than the diameter of the arterioles alone. The identity of the tyrosine-phosphorylated proteins requires further investigation; however, there is some evidence supporting roles for cSrc-type tyrosine kinases and p44 mitogen-activated protein kinase. The longer-term responses of blood vessels to increased transmural pressure that may involve tyrosine phosphorylation pathways include maintenance of myogenic constriction and vessel wall remodelling.

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