Biomedical subjects

Tudor M Griffith

Publications and source records attributed to Tudor M Griffith.

15 recordsLinked to original sources

PubMed2006

Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries.

Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ -sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.

Animals↗

PubMed2005

5-Methyltetrahydrofolate and tetrahydrobiopterin can modulate electrotonically mediated endothelium-dependent vascular relaxation.

We have investigated the ability of 5-methyltetrahydrofolate (5-MTHF) and tetrahydrobiopterin (BH(4)) to modulate nitric oxide (NO)-independent vascular relaxations that are mediated by the sequential spread of endothelial hyperpolarization through the wall of the rabbit iliac artery by means of myoendothelial and homocellular smooth muscle gap junctions. Relaxations and subintimal smooth muscle hyperpolarizations evoked by cyclopiazonic acid were depressed by the gap junction inhibitor 2-aminoethoxydiphenyl borate, whose effects were prevented by 5-MTHF and BH(4), but not by their oxidized forms folic acid and 7,8-dihydrobiopterin. Analogously, 5-MTHF and BH(4), but not folic acid or 7,8-dihydrobiopterin, attenuated the depression of subintimal hyperpolarization by a connexin-mimetic peptide targeted against Cx37 and Cx40 ((37,40)Gap 26) and the depression of subadventitial hyperpolarization by a peptide targeted against Cx43 ((43)Gap 26), thus reflecting the known differential expression of Cx37 and Cx40 in the endothelium and Cx43 in the media of the rabbit iliac artery. The inhibitory effects of 2-aminoethoxydiphenyl borate and (37,40)Gap 26 against subintimal hyperpolarization were prevented by catalase, which destroys H(2)O(2). 5-MTHF and BH(4) thus appear capable of modulating electrotonic signaling by means of myoendothelial and smooth muscle gap junctions by reducing oxidant stress, potentially conferring an ability to reverse the endothelial dysfunction found in disease states through mechanisms that are independent of NO.

Animals↗

PubMed2005

Connexin-mimetic peptides dissociate electrotonic EDHF-type signalling via myoendothelial and smooth muscle gap junctions in the rabbit iliac artery.

Synthetic peptides corresponding to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40 and Cx43), designated as (43)Gap 26, (40)Gap 27, (37,40)Gap 26 and (37,43)Gap 27 according to Cx homology, were used to investigate the role of gap junctions in the spread of endothelial hyperpolarizations evoked by cyclopiazonic acid (CPA) through the wall of the rabbit iliac artery. Immunostaining and confocal microscopy demonstrated that gap junction plaques constructed from Cx37 and Cx40 were abundant in the endothelium, whereas Cx43 was the dominant Cx visualized in the media. None of the Cx-mimetic peptides affected endothelial hyperpolarizations evoked by CPA directly. When administered individually, (40)Gap 27, (37,40)Gap 26 and (37,43)Gap 27, but not (43)Gap 26, attenuated endothelium-dependent subintimal smooth muscle hyperpolarization. By contrast, only (43)Gap 26 and (37,43)Gap 27 reduced the spread of subintimal hyperpolarization through the media of the rabbit iliac artery. The site of action of the peptides therefore correlated closely with the expression of their target Cxs in detectable gap junction plaques. The findings provide further evidence that the EDHF phenomenon is electrotonic in nature, and highlight the contribution of myoendothelial and homocellular smooth muscle communication via gap junctions to arterial function.

Animals↗

PubMed2005

Effects of connexin-mimetic peptides on gap junction functionality and connexin expression in cultured vascular cells.

1. We have investigated the effects of connexin-mimetic peptides homologous to the Gap 26 and Gap 27 domains of Cxs 37, 40 and 43 against gap junctional communication and connexin expression in rat aortic endothelial cells (RAECs) and A7r5 myocytes. 2. Immunostaining and Western blot analysis confirmed the presence of gap junction plaques containing Cx43, but not Cx40, in RAECs, whereas plaques containing Cxs 40 and 43 were evident in A7r5 cells. Expression of Cx37 was limited in RAECs and absent from A7r5 cells. 3. Under control conditions calcein-loaded RAECs transferred dye to approximately 70% of subjacent A7r5 cells after coculture for 4-5 h. Dye transfer was inhibited by a peptide targeted to Cxs 37 and 43 ((37,43)Gap 27), but minimally affected by peptides targeted to Cxs 37 and 40 ((37,40)Gap 26 and (40)Gap 27). These findings suggest that the myoendothelial gap junctions that couple RAECs and A7r5 cells are constructed principally from Cx43. 4. Inhibition of dye transfer from RAECs to A7r5 cells cocultured in the presence of (37,43)Gap 27 plus (37,40)Gap 26 for 5 h was fully reversible. 5. In A7r5 cells, endogenous expression of Cx40 and Cx43 was unaffected by incubation with (37,43)Gap 27, (37,40)Gap 26, either individually or in combination, and the peptide combination did not impair connexin trafficking or the de novo formation of gap plaques in A7r5 cells transfected to express Cx43-GFP. 6. Treatment of A7r5 cells with (37,43)Gap 27 plus (37,40)Gap 26 abolished synchronized oscillations in intracellular [Ca2+] induced by the alpha1-adrenoceptor agonist phenylephrine. 7. The reversibility and lack of effect of the peptides on plaque formation suggests that they may be considered ideal probes for functional studies of connexin-mediated communication in the vascular wall.

Animals↗

PubMed2004

The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization.

Although an endothelium-derived hyperpolarizing factor (EDHF) has often been hypothesized to underpin vascular relaxations that are independent of nitric oxide (NO) and prostanoids, bioassay techniques have failed to confirm the existence of a freely transferable EDHF in a consistent fashion. Indeed, observations that inhibitors of direct cell-cell coupling such as connexin-mimetic peptides (e.g. Gap 26 and 27) and glycyrrhetinic acid derivatives attenuate "EDHF-type" smooth muscle hyperpolarizations and relaxations suggest that an electrotonic spread of endothelial hyperpolarization via myoendothelial and homocellular smooth muscle gap junctions plays an obligatory role in such responses. The endothelial hyperpolarization that initiates relaxation results from the opening of K(Ca) channels and is sustained by capacitative Ca(2+) entry triggered by the depletion of intracellular Ca(2+) stores in the endoplasmic reticulum. EDHF-type relaxations are also associated with a prostanoid-independent synthesis of cAMP that increases the conductance of gap junction channels and enhances the transmission of endothelial hyperpolarization through the vascular wall in a permissive fashion. This review will discuss the roles of these interacting signalling pathways in the mediation of the EDHF phenomenon.

Amino Acid Sequence↗

PubMed2004

Endothelium-dependent smooth muscle hyperpolarization: do gap junctions provide a unifying hypothesis?

An endothelium-derived hyperpolarizing factor (EDHF) that is distinct from nitric oxide (NO) and prostanoids has been widely hypothesized to hyperpolarize and relax vascular smooth muscle following stimulation of the endothelium by agonists. Candidates as diverse as K(+) ions, eicosanoids, hydrogen peroxide and C-type natriuretic peptide have been implicated as the putative mediator, but none has emerged as a 'universal EDHF'. An alternative explanation for the EDHF phenomenon is that direct intercellular communication via gap junctions allows passive spread of agonist-induced endothelial hyperpolarization through the vessel wall. In some arteries, eicosanoids and K(+) ions may themselves initiate a conducted endothelial hyperpolarization, thus suggesting that electrotonic signalling may represent a general mechanism through which the endothelium participates in the regulation of vascular tone.

Animals↗

PubMed2004

Enhanced inhibition of the EDHF phenomenon by a phenyl methoxyalaninyl phosphoramidate derivative of dideoxyadenosine.

In rabbit arteries endogenous production of cAMP facilitates electrotonic signalling via gap junctions, thus explaining the ability of P-site inhibitors of adenylyl cyclase to attenuate EDHF-type responses. In the present study, we show that a lipophilic phosphoramidate pronucleotide derivative of dideoxyadenosine, 2',3'-ddA-PMAPh, exhibits enhanced activity as an inhibitor of EDHF-type smooth muscle hyperpolarizations induced by acetylcholine (ACh) compared to the parent nucleoside 2',3'-ddA, and that the effects of both compounds can be reversed by the cAMP phosphodiesterase inhibitor IBMX. Neither 2',3'-ddA nor 2',3'-ddA-PMAPh depress ACh-evoked endothelial hyperpolarization directly. Modifications in the lipophilicity of dideoxyadenosine and its direct intracellular delivery as a mononucleotide may thus enhance the ability to inhibit adenylyl cyclase and depress electrotonic signalling via myoendothelial gap junctions.

Amides↗

PubMed2003

Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries.

We have compared the contributions of gap junctional communication and chemical signaling via H2O2 to NO-independent relaxations evoked by the Ca2+ ionophore A23187 and acetylcholine (ACh) in rabbit ilio-femoral arteries. Immunostaining confirmed the presence of connexins (Cxs) 37 and 40 in the endothelium and Cxs 40 and 43 in smooth muscle. Maximal endothelium-dependent subintimal smooth muscle hyperpolarizations evoked by A23187 and ACh were equivalent (approximately 20 mV) and almost abolished by an inhibitory peptide combination targeted against Cxs 37, 40, and 43. However, maximal NO-independent relaxations evoked by A23187 were unaffected by such peptides, whereas those evoked by ACh were depressed by approximately 70%. By contrast, the enzyme catalase, which destroys H2O2, attenuated A23187-induced relaxations over a broad range of concentrations, but only minimally depressed the maximum response to ACh. Catalase did not affect A23187- or ACh-evoked hyperpolarizations. After loading with an H2O2-sensitive probe, A23187 caused a marked increase in endothelial fluorescence that correlated temporally with relaxation, whereas only a weak delayed increase was observed with ACh. In arteries without endothelium, the H2O2-generating system xanthine/xanthine oxidase induced a catalase-sensitive relaxation that mimicked the gap junction-independent response to A23187 as it was maximally equivalent to approximately 80% of induced tone, but associated with a smooth muscle hyperpolarization <5 mV. We conclude that myoendothelial gap junctions underpin smooth muscle hyperpolarizations evoked by A23187 and ACh, but that A23187-induced relaxation is dominated by extracellular release of H2O2. Endothelium-derived H2O2 may thus be regarded as a relaxing factor, but not a hyperpolarizing factor, in rabbit arteries.

Acetylcholine↗

PubMed2003

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed approximately 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Animals↗

PubMed2003

Essential role of Gap junctions in NO- and prostanoid-independent relaxations evoked by acetylcholine in rabbit intracerebral arteries.

BACKGROUND AND PURPOSE: Direct intercellular communication via gap junctions may play a central role in endothelium-dependent relaxations that are mediated by a conducted hyperpolarization and do not involve the synthesis of NO and prostanoids. In the present study, inhibitory peptides homologous to the Gap27 domain of the second extracellular loop of connexin37/connexin43 and connexin40, designated as 37,43Gap27 and 40Gap27, respectively, were used to evaluate the role of this mechanism in intracerebral arteries. METHODS: Isolated rings of rabbit middle cerebral artery were constricted by histamine (10 micromol/L) in the presence of N(G)-nitro-L-arginine methyl ester (300 micromol/L) and indomethacin (10 micromol/L). Concentration-relaxation curves for acetylcholine were constructed in the presence and absence of 37,43Gap27 and 40Gap27. Specific antibodies were used to delineate the distribution of connexin37, connexin40, connexin43, and connexin45 within the arterial wall. RESULTS: Individually, 37,43Gap27 and 40Gap27 minimally affected endothelium-dependent relaxations to acetylcholine at concentrations of 300 micro mol/L, whereas their combination (at 300 micromol/L each) inhibited the maximal response by approximately 70% and increased the EC50 value for relaxation by approximately 15-fold. In endothelium-denuded rings, this peptide combination did not attenuate responses to sodium nitroprusside, an exogenous source of NO. Gap junction plaques, whose incidence was highest in endothelium, were constructed from connexin40 and connexin43 in the media and connexin37, connexin40, and connexin43 in the endothelium. CONCLUSIONS: The findings confirm that direct communication via gap junctions contributes to agonist-induced relaxations of intracerebral arteries. More than one connexin subtype appears to participate in such responses.

Acetylcholine↗

PubMed2002

cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions.

We have investigated the role of cAMP in NO- and prostanoid-independent relaxations that are widely attributed to an endothelium-derived hyperpolarizing factor (EDHF). Under control conditions EDHF-type relaxations evoked by acetylcholine (ACh) in rabbit iliac arteries were transient, but in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the cell permeant cAMP analog 8-bromo-cAMP, relaxations became sustained with their maxima potentiated approximately 2-fold. Relaxation was associated with transient approximately 1.5-fold elevations in smooth muscle cAMP levels with both mechanical and nucleotide responses being abolished by interrupting gap junctional communication with the connexin-mimetic peptide Gap 27 and by endothelial denudation. However, IBMX induced a sustained endothelium-independent approximately 2-fold rise in cAMP levels, which was not further amplified by ACh, suggesting that the contribution of cAMP to the EDHF phenomenon is permissive. After selective loading of the endothelium with calcein AM, direct transfer of dye from the endothelium to the media was enhanced by IBMX or 8-bromo-cAMP, but not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration within the intima. ACh-induced hyperpolarizations of subintimal smooth muscle in arterial strips with intact endothelium were abolished by Gap 27 and the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine but were unaffected by IBMX. By contrast, in strips partially denuded of endothelium, IBMX enhanced the transmission of hyperpolarization from the endothelium to remote smooth muscle cells. These findings support the hypothesis that endothelial hyperpolarization underpins the EDHF phenomenon, with cAMP governing subsequent electrotonic signaling via both myoendothelial and homocellular smooth muscle gap junctions.

Animals↗

PubMed2002

Relative contributions of NO and gap junctional communication to endothelium-dependent relaxations of rabbit resistance arteries vary with vessel size.

Two synthetic peptide inhibitors of gap junctional communication have been used to compare the contribution of direct cell-cell coupling to acetylcholine-induced relaxations of the rabbit central ear artery (G(0)) and its second branch generation (G(2)). These peptides, designated (43)Gap 26 and (37,43)Gap 27, possess sequence homology with specific domains of the first extracellular loop of connexin 43 (Cx43) and second extracellular loop of Cxs 37 and 43, respectively. Immunohistochemistry confirmed the presence of Cxs 37, 40, and 43 in the vascular endothelium, but of only Cx43 in the media of G(0). At concentrations of 300 microM, (43)Gap 26 and (37,43)Gap 27 each inhibited the maximum response to acetylcholine in G(2) by approximately 50%, but by only approximately 20% in G(0), whereas inhibition of NO synthesis by 300 microM N(G)-nitro-L-arginine methyl ester attenuated maximum relaxations to acetylcholine by approximately 30% in G(2), but by approximately 70% in G(0). Residual endothelium-derived hyperpolanizing factor-type responses in G(0) and G(2) were abolished by (43)Gap 26 and (37,43)Gap 27. In HeLa cells transfected to express a chimeric Cx43-green fluorescent protein that forms functional gap junctions, the peptides were equally effective inhibitors of Lucifer yellow dye transfer. We conclude that the contribution of gap junctions to endothelium-dependent relaxation is inversely related to vessel size and exhibits an apparently reciprocal relationship with NO-mediated mechanisms of vasorelaxation in the rabbit ear.

Acetylcholine↗

PubMed2002

Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation.

We have compared the mechanisms that contribute to endothelium-derived hyperpolarizing factor (EDHF)-type responses induced by ACh and the Ca(2+) ionophore A-23187 in the rabbit iliac artery. Relaxations to both agents were associated with ~1.5-fold elevations in smooth muscle cAMP levels and were attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA) and potentiated by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Mechanical responses were inhibited by coadministration of the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin, both in the absence and presence of IBMX, but were unaffected by blockade of ATP-sensitive K(+) channels with the sulphonylurea glibenclamide. Relaxations and elevations in cAMP evoked by ACh were abolished by 18alpha-glycyrrhetinic acid, which disrupts gap junction plaques, whereas the corresponding responses to A-23187 were unaffected by this agent. Consistently, in "sandwich" bioassay experiments, A-23187, but not ACh, elicited extracellular release of a factor that evoked relaxations that were inhibited by DDA and potentiated by IBMX. These findings provide evidence that EDHF-type relaxations of rabbit iliac arteries evoked by ACh and A-23187 depend on cAMP accumulation in smooth muscle, but involve signaling via myoendothelial gap junctions and the extracellular space, respectively.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Endogenous nitric oxide synthesis differentially modulates pressure-flow and pressure-conductance relationships in the internal and external carotid artery circulations of the rat.

The role of endogenous nitric oxide (NO) synthesis was investigated in the regulation of the internal (ICA) and external carotid artery (ECA) beds of ventilated, anesthetized rats in a model in which the left common carotid artery was perfused from the aorta via an extracorporeal circuit under conditions of non-pulsatile controlled flow. The territories supplied by the extracranial ICA and ECA were studied separately following occlusion of the appropriate artery. An inhibitor of nitric oxide synthesis, N(G)-monomethyl-L-arginine (L-NMMA), and the NO synthase substrate L-arginine were administered via a jugular venous catheter. NO synthesis exerted an important influence on the pressure-flow relationships of the ICA and ECA circulations as L-NMMA increased input perfusion pressure at any given flow rate. However, in the presence of NO synthesis, hydraulic conductance increased rapidly with flow in the ICA, thereby stabilizing perfusion pressures over a wide range of flow rates, whereas this phenomenon was not evident in the ECA territory. Differences between the two circulations were further emphasized by observations that L-arginine antagonized the systemic hemodynamic response to L-NMMA and its effects on the conductance of the ECA bed, whereas the effects of L-NMMA were irreversible in the ICA territory.

Animals↗

PubMed2002

Endothelial control of vascular tone by nitric oxide and gap junctions: a haemodynamic perspective.

Local haemodynamic forces acting on the endothelium modulate vascular tone through mechanisms that normalize intimal shear stress. This flow-dependent diameter response contributes to the optimization of circulatory function and is mediated via shear stress-induced release of NO, vasodilator prostanoids and a putative endothelium-derived hyperpolarizing factor or EDHF. There is growing evidence that NO/prostanoid independent relaxations involve direct heterocellular signalling between endothelial and smooth muscle cells via gap junctions.

Animals↗