Biomedical subjects
Publications and source records attributed to A D Hughes.
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We examined the effect of platelet-derived growth factor (PDGF)-AA, PDGF-AB, and PDGF-BB isoforms on DNA synthesis, Ca2+ mobilization, and tyrosine phosphorylation in cultured human saphenous vein cells cultured by an explant technique; confluent cells derived from passage 3 were used for all studies. DNA synthesis was measured by [methyl3H]thymidine uptake, intracellular [Ca2+] ([Ca2+]i) was measured with the Ca(2+)-sensitive indicator fura-2, and tyrosine phosphorylation was measured by Western blotting techniques. All three isoforms of PDGF stimulated [methyl3H]thymidine uptake concentration dependently, with similar potency. PDGF-AB induced significantly greater [methyl3H]-thymidine uptake than PDGF-BB, and PDGF-AA was much less effective than either PDGF-AB or PDGF-BB. The effects of all three isoforms were inhibited by tyrphostin-23, a selective inhibitor of tyrosine kinases PDGF-AB and PDGF-BB increased [Ca2+]i, although the maximum response to PDGF-AB was significantly less than that to PDGF-BB. Both isoforms increased [Ca2+]i by stimulating influx and intracellular release of Ca2+. PDGF-AA had no measurable effect on [Ca2+]i. All three isoforms increased tyrosine phosphorylation of a 170-kDa protein detected by Western blotting. Quantitative densitometry indicated that PDGF-BB induced greater tyrosine phosphorylation than PDGF-AB and both PDGF-BB and PDGF-AB induced markedly more tyrosine phosphorylation than PDGF-AA. PDGF isoforms have differing efficacies in terms of DNA synthesis, Ca2+ mobilization, and tyrosine phosphorylation by human saphenous vein cells.
We examined the effects of ryanodine and thapsigargin on changes in cytoplasmic [Ca2+] (Cai) and muscle tension in rat mesenteric resistance arteries induced by norepinephrine (NE) and caffeine. Both ryanodine and thapsigargin markedly inhibited the increase in Cai and contractile responses to caffeine in physiological saline and to NE and caffeine in calcium-free conditions. In contrast, peak responses to potassium depolarisation and NE in physiological saline appeared little affected, although time taken to achieve 50% of peak response after addition of NE was slowed after ryanodine and thapsigargin treatment. Neither ryanodine nor thapsigargin altered resting tone or Cai or the Ca2+ sensitivity of contraction under depolarized conditions. The NE concentration-response relationship was not significantly altered after ryanodine or thapsigargin. Ryanodine and thapsigargin inhibit the release of intracellular Ca2+ stores by NE and caffeine. Inhibition of release of intracellular Ca2+ by NE has only slight effects on contractile responses of mesenteric resistance arteries when extracellular Ca2+ is present.
1. The effect of platelet-derived growth factor (PDGF-AB) on tone and intracellular Ca2+ ([Ca2+]i) was examined in rabbit isolated ear arteries. Arteries were mounted in a myograph and loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2, for concurrent measurements of isometric force and [Ca2+]i. 2. PDGF-AB contracted rabbit ear artery in a concentration-dependent manner. PDGF-AB induced tone was associated with a rise in [Ca2+]i. In the presence of noradrenaline, PDGF-AB induced a similar rise in [Ca2+]i but contraction in response to PDGF-AB in the presence of noradrenaline was increased compared with PDGF-AB alone. 3. PDGF-AB-induced rise in [Ca2+]i and tone were abolished by removal of extracellular Ca2+ (with addition of BAPTA, a Ca2+ chelator), and by preincubation with a dihydropyridine calcium channel blocker, (-)-202,791. Bistyrphostin, a selective inhibitor of tyrosine kinases, also inhibited PDGF-AB-induced tone, but had no effect on noradrenaline- or potassium-induced tone. 4. PDGF-AB contracts rabbit ear artery by increasing Ca2+ entry through voltage-operated calcium channels. This effect involves activation of a tyrosine kinase.
1. Platelet derived growth factor (PDGF), AB and BB isoforms (100 pM) increased calcium channel currents measured by whole cell voltage clamp technique in single vascular smooth muscle cells isolated from rabbit ear arteries. 2. Tyrphostin-23 (100 microM) a selective inhibitor of protein tyrosine kinases, reduced calcium channel currents. Pre-incubation with tyrphostin-23 prevented PDGF-AB induced increase in calcium channel currents. However, in these same cells 10 nM (+)-202791, a dihydropyridine calcium channel agonist, did increase calcium channel currents. 3. Bistyrphostin (10 microM), a selective inhibitor of epidermal growth factor (EGF)-kinase also reduced calcium channel currents and inhibited PDGF-AB-induced increases in calcium channel currents. 4. Genistein (100 microM) a selective inhibitor of tyrosine kinases, structurally unrelated to the tryphostins, also inhibited calcium channel currents and pre-incubation with genistein prevented the PDGF-AB-induced rise in calcium channel currents. 5. These results indicate that PDGF increases calcium channel currents in vascular smooth muscle. This action of PDGF probably involves a tyrosine kinase.
1. We examined the effect of the thiazide diuretic, hydrochlorothiazide, on on intracellular calcium concentration ([Ca2+]i) and tone in guinea-pig mesentery arteries. Vessels were mounted on a microvascular myograph and loaded with the Ca(2+)-sensitive fluorescent dye, Fura-2. 2. Hydrochlorothiazide caused relaxation of noradrenaline-precontracted arteries associated with a fall in [Ca2+]i. Preincubation of arteries with hydrochlorothiazide inhibited both contraction and rise in [Ca2+]i in response to noradrenaline. Hydrochlorothiazide did not affect tone and [Ca2+]i when this was elevated by a combination of depolarizing potassium solution and noradrenaline. 3. Hydrochlorothiazide-induced vasorelaxation and decrease of [Ca2+]i was abolished by charybdotoxin, a blocker of large conductance Ca(2+)-activated K channels. 4. The rise in [Ca2+]i elicited by caffeine in Ca(2+)-free physiological salt solution, and presumably reflecting Ca2+ release from intracellular stores, was not altered by preincubation with hydrochlorothiazide. 5. Under depolarizing conditions hydrochlorothiazide did not alter the relationship between the extracellular concentration of Ca2+ and [Ca2+]i; however, hydrochlorothiazide caused a small reduction in the contraction produced for a given rise in [Ca2+]i suggesting hydrochlorothiazide may cause a slight desensitization of the contractile machinery. 6. These findings suggest that hydrochlorothiazide opens Ca(2+)-activated K channels leading to hyperpolarization and consequent closing of voltage-operated calcium channels. The result of this is an impaired influx of extracellular Ca2+, a decrease in [Ca2+]i and vasorelaxation.
Arterial hypertension is associated with structural changes in the cardiovascular system. This study has examined the effect of hypertension on the carotid artery wall and examined the relation between changes in the structure of carotid artery wall and left ventricle in untreated hypertensives. The carotid artery wall was visualized using a high resolution ultrasound technique in 37 untreated hypertensive patients (25 males, 12 females) and 37 age and sex matched normotensive individuals and carotid intima-media thickness (IMT) and carotid artery diameter measured. IMT and intima-media cross sectional area was significantly greater in the hypertensive group compared with the normotensive group, though the carotid artery diameter did not differ significantly. There was a significant association between age and IMT in both groups. In the hypertensive group there was also a significant association between left ventricular mass index, ventricular septal or posterior wall thickness and IMT. This study indicates that there is an association between cardiac and carotid arterial structure in hypertension. Such a relationship may be important in understanding the associated risks of high blood pressure.
BACKGROUND: Cardiac assessment is an important part of risk stratification in hypertensive patients. Left ventricular hypertrophy in particular is a powerful predictor of subsequent cardiovascular morbidity and mortality. Previous studies assessing haemodynamic factors that may be responsible for cardiac changes in hypertensive patients have been performed in those previously treated for hypertension. To investigate more fully these haemodynamic relationships, a large group of previously untreated patients were studied. METHODS: Ninety-eight previously untreated hypertensive patients underwent electrocardiography, two-dimensional and Doppler echocardiography, 24 h ambulatory blood pressure monitoring and exercise stress testing. RESULTS: The left ventricular mass index (LVMI) was more closely related to mean 24 h than to clinic blood pressures (24 h systolic r = 0.48, P < 0.01; 24 h diastolic r = 0.49, P < 0.01; clinic systolic r = 0.28, P < 0.01; clinic diastolic r = 0.31, P < 0.01). In addition, the systolic nocturnal blood pressure dip was found to be inversely related to LVMI in men (r = -0.32, P < 0.01). Of the indices of left ventricular diastolic function, age (r = -0.64, P < 0.01), heart rate (r = -0.25, P = 0.02) and LVMI (r = -0.22, P = 0.02) were independently related to the E-A ratio. Age (r = 0.40, P < 0.01), blood pressure (systolic r = 0.39, P < 0.01; diastolic r = 0.43, P < 0.01), the nocturnal blood pressure dip (systolic r = -0.38, P < 0.01, diastolic r = -0.31, P < 0.01) and LVMI (r = 0.37, P < 0.01) were independently related to the isovolumic relaxation time. CONCLUSIONS: Blood pressure was the only independent determinant of LVMI; nocturnal blood pressure may be particularly important in men. Age and both haemodynamic and structural factors are independent determinants of parameters of left ventricular diastolic function in hypertensive patients.
OBJECTIVE: The aim was to investigate the involvement of polyamines in the development of cardiovascular hypertrophy in a rat angiotensin II infusion model of hypertension. METHODS: Rats were chronically infused with a low dose of angiotensin II (83 ng.min-1.rat-1) or vehicle by osmotic minipumps. The polyamine content of the cardiovascular organs and liver was measured by HPLC, and the blood pressure of conscious rats was determined by a tail cuff method. The mesenteric arterial beds were perfusion fixed in situ and medial and luminal cross sectional areas were determined by computer assisted planimetry. Cardiac hypertrophy was assessed by determination of ventricular:body weight ratios. RESULTS: Angiotensin II infusion caused an increased concentration of polyamines in the ventricles and aorta within hours of beginning the infusion, and within days in the mesenteric arteries. Polyamine content of the liver was unaffected. Hypertrophy of the myocardium and mesenteric arteries in association with the increased blood pressure was evident. CONCLUSIONS: A low dose of angiotensin II stimulates an early biochemical marker of growth, the polyamines, in cardiovascular tissues but not in the liver. The increase in tissue polyamines is associated with cardiovascular hypertrophy and the development of hypertension.
Heparin and ruthenium red were introduced intracellularly into rat mesenteric resistance arteries via reversible-permeabilisation. Heparin and ruthenium red inhibited contractile responses to noradrenaline, but not caffeine in Ca(2+)-free conditions. Neither heparin nor ruthenium red significantly inhibited peak contractile responses to K+, noradrenaline or caffeine in physiological saline, although heparin significantly increased the time taken for peak force to develop in response to noradrenaline. Noradrenaline and calcium concentration-response relationships were unaffected by heparin. Experiments with permeabilised, fura-2 loaded vessels indicated that heparin inhibited Ca2+ release induced by noradrenaline, but did not inhibit caffeine-induced Ca2+ release. The peak rise in intracellular Ca2+ following K+, or noradrenaline in physiological saline was unaffected by heparin. The use of reversible permeabilisation may prove a useful approach, allowing introduction of a variety of membrane-impermeant blockers of second messenger systems into intact resistance arteries.
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The actions of the optical enantiomers of BAY K 8644 and Sandoz 202,791 were studied on barium inward currents recorded using the whole-cell configuration of the patch clamp technique from enzymatically isolated smooth muscle cells from the rabbit ear artery. The enantiomers were applied by bath perfusion or rapidly by a concentration jump technique, which enabled the study of drug action under equilibrium and nonequilibrium conditions. A larger effect of agonists was seen on peak inward current in 110 mM Ba when small rather than large depolarizations were applied. The midpoint voltage of the steady-state inactivation curve of IBa was -12.8 +/- 1.9 mV (n = 4) in the absence of drug, -16.4 +/- 2.5 mV (n = 4) in 1 microM (+)202,791, and -31.4 +/- 0.4 mV (n = 4) in 1 microM (-)202,791. The rate of onset of action of the agonist and antagonist enantiomers of BAY K 8644 and Sandoz 202,791 was studied by rapid application during 20-ms depolarizing steps from different holding potentials to +30 mV at 1 or 0.2 Hz. The drugs were applied as concentration jumps between two single pulses of a pulse train. The rates of onset of drug action on peak IBa during a 1-Hz pulse train were concentration dependent over the range of 100 nM-3 microM for both (+) and (-)202,791. The rate of onset of inhibition of peak current by antagonist enantiomers was not significantly influenced by the test pulse frequency. At a holding potential of -60 mV, the onset rate of the increase in peak IBa on application of 1 microM of agonist enantiomers (+)202,791 or (-)BAY K 8644 during a train of pulses occurred with mean time constants of 2.1 +/- 0.7 s (n = 7) and 2.3 +/- 0.2 s (n = 4), respectively. The onset of current increase on application of 1 microM (+)202,791 during a single voltage clamp step to 20 mV was faster, with a mean time constant of 380 +/- 80 ms (n = 3).
Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.
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1. Human subcutaneous resistance arteries (internal diameter 113-626 microns) were mounted in an isometric myograph. Electrical field stimulation was applied either continuously in the form of a frequency-response curve or intermittently at 16 Hz. The magnitude of the maximum contraction induced by continuous stimulation expressed as a percentage of the response to a supramaximal concentration of noradrenaline (10 microM) was highly variable but unrelated to vessel calibre. Contractile responses to both continuous and intermittent stimulation were abolished by 1 microM tetrodotoxin. 2. Prazosin (100 nM and 1 microM, alpha 1-adrenoceptor antagonist) inhibited responses to continuous stimulation over a range of frequencies (2-8 Hz). The response to continuous stimulation at 8 Hz was inhibited by 78 +/- 6% by 1 microM prazosin. Rauwolscine (100 nM, alpha 2-adrenoceptor antagonist) had a smaller effect on contractions induced by continuous stimulation. Rauwolscine inhibited the response at 8 Hz by 36 +/- 6%. Rauwolscine at a higher concentration (1 microM) caused further inhibition of the response to continuous stimulation. Prazosin and rauwolscine in combination almost completely inhibited the response to continuous stimulation at concentrations of 1 microM. 3. Prazosin and rauwolscine inhibited responses to intermittent stimulation in a concentration-dependent manner. The IC50 for this action of prazosin was 3.7 +/- 1.6 nM and the maximum inhibition induced by 100 nM prazosin was 78 +/- 6%. The IC50 of rauwolscine was 12.0 +/- 1.3 nM and 100 nM rauwolscine caused a 86 +/- 7% reduction in the response to intermittent stimulation.Prazosin and rauwolscine in combination (each at 100 nM) caused marked inhibition of the response to intermittent stimulation leaving only 7.0 +/- 2.6% of the response.4. These data suggest that neurally released noradrenaline evokes contractions of human resistance arteries by activation of both alpha 1,- and alpha 2-adrenoceptors postjunctionally.
The effect of caffeine on inward current carried by barium ions through voltage-dependent calcium channels has been investigated in single rabbit ear artery cells using whole-cell voltage-clamp techniques. Caffeine (1-30 mM) caused a rapid and reversible concentration-dependent blockade of barium current and a related compound, 3-isobutyl-1-methylxanthine (IBMX), was a more potent inhibitor of barium current. Caffeine-induced inhibition of barium current showed no voltage- or use-dependence and caffeine did not alter the steady-state inactivation of barium current. The effect of caffeine was not blocked by extracellular or by intracellular ryanodine or inclusion of both 5 mM 1,2-bis(2-aminophenoxy)-ethane N,N,N',N',-tetraacetic acid (BAPTA) and 2 mM ethylene glycol-bis(beta-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) in the intracellular solution. Rolipram and M&B 22984, non-xanthine inhibitors of phosphodiesterase, did not diminish inward barium current. The data indicate that caffeine and IBMX block voltage-operated calcium channels and it is suggested that this is due to a direct interaction of methylxanthines with the calcium channel.
S(+)-PN 202-791, a calcium channel agonist, and its optical isomer R(-)-PN 202-791, a calcium channel antagonist, respectively increased and decreased inward current carried by barium through voltage-dependent calcium channels in isolated ear artery cells of the rabbit in a concentration-dependent manner. The EC50 or IC50 derived from the concentration-response relationship was unaltered in the presence of the other enantiomer indicating that no competitive antagonism exists between these enantiomers and suggesting that the actions of these two enantiomers involve two separate binding sites.