Linda Shaw

Identification of Protein Kinase C Isoforms in Rat Mesenteric Small Arteries and Their Possible Role in Agonist-Induced Contraction

We have identified immunologically the protein kinase C (PKC) isoforms present in rat mesenteric small arteries, defined their distribution between particulate and soluble fractions, and studied their involvement in phorbol ester-induced contraction. Our analysis revealed the presence of the CA(2+)-dependent PKCs (alpha and gamma), Ca(2+)-independent PKCs (delta and epsilon), and the atypical isoform (zeta). PKCbeta could not be detected, whereas PKCgamma is likely to be of neural origin. All isoforms exhibited different distributions. PKCalpha, PKCepsilon, and PKCzeta were found in both particulate and soluble fractions. In contrast, PKCdelta was mainly in the particulate fraction, and PKCgamma was in the soluble fraction. Phorbol esters, which activate PKC and cause smooth muscle contraction, downregulated only the alpha and delta isoforms. This was associated with a parallel loss of contractile response to phorbol ester. The force developed to submaximal concentrations of noradrenaline was decreased after phorbol dibutyrate pretreatment, although the sensitivity and maximal response were unchanged. Phorbol ester pretreatment did not affect the contractile response to vasopressin. The sensitivity to non-receptor-mediated contraction, caused by k+ in the presence of prazosin, was slightly reduced by 4 alpha- and 4 beta-phorbol ester pretreatment. Maximal tension in response to this agonist was not affected. We conclude that PKCalpha and/or PKCdelta is necessary for phorbol ester-mediated contraction but is not essential for noradrenaline-, vasopressin-, or k(+)-induced contraction, demonstrating differences in the mechanisms involved in the contractile response between these agents.

Mechanisms of 17 β-oestradiol induced vasodilatation in isolated pressurized rat small arteries

The influence of 17 β‐oestradiol on pressurized isolated rat mesenteric and coronary small arteries was investigated. 17 β‐oestradiol caused rapid (t1.0<5 mins) concentration‐dependent relaxations of pre‐contracted pressurized (50 mmHg) isolated rat mesenteric and coronary arteries. Similar responses were observed in both vessel types. Significant relaxations were only observed at concentrations exceeding 3 μM. The vasodilatory responses in both types of artery were unaffected by 10 μM L‐nitro arginine (L‐NNA) alone or in the presence of 10 μM indomethacin, inhibitors of nitric oxide and prostaglandin synthesis respectively. They were also unaffected by the pre‐contracting agent used i.e. high K+ or U46619 (a thromboxane analogue). Neither the oestrogen receptor antagonist ICI 182,780 (10 μM) nor the protein synthesis inhibitor cycloheximide (100 μM) had any effect on the responses of mesenteric arteries to 17 β‐oestradiol. 17 α‐oestradiol had only a minor effect on mesenteric arterial diameter over a concentration range similar to the effective vasodilatory range for 17 β‐oestradiol. Membrane impermeant 17 β‐oestradiol conjugated to bovine serum albumin (β‐oestradiol‐17hemisuccinate‐BSA) (E‐H‐BSA) resulted in a vasodilatation of pressurized arteries. Wortmannin, an inhibitor of myosin light chain kinase, near maximally relaxed pressurized mesenteric arteries although the time course for the response was significantly slower than that for 17 β‐oestradiol. These results taken together suggest that the acute effects of 17 β‐oestradiol on isolated pressurized arterial tone may be due to effects directly on the vascular smooth muscle via non‐genomic mechanisms that involve a stereospecific interaction at the plasma membrane.

αV Integrins Are Necessary for Eutrophic Inward Remodeling of Small Arteries in Hypertension

Human essential hypertension is characterized by eutrophic remodeling of small arteries, with little evidence of hypertrophy. Likewise, vessels of young hypertensive TGR(mRen2)27 animals have undergone similar structural alterations. The role of integrins in resistance arteries of TGR(mRen2)27 during the eutrophic-remodeling process was examined as blood pressure rose. Initially, 8 &agr; and 3 &bgr; integrins were identified and levels of expression investigated using RT-PCR. As pressure increased and remodeling advanced, integrin expression profiles revealed that only &agr;V was significantly raised. In conjunction, we confirmed elevated integrin &agr;V protein levels in TGR(mRen2)27 rat arteries and localization to the media using immunofluorescence. &bgr;1 and &bgr;3, but not &bgr;5 integrin subunits were coprecipitated with integrin &agr;V and are implicated in the eutrophic remodeling process. Administration of a peptide antagonist of &agr;V&bgr;3 abolished remodeling but enhanced growth, indicating that hypertrophy supervened as a response to hypertension-induced increases in wall stress. We have established that the only upregulated integrin, the &agr;V subunit of integrin &agr;V&bgr;3, has a crucial role in the hypertensive remodeling process of TGR(mRen2)27 rat resistance arteries. During hypertensive remodeling, functions of specific &agr;V&bgr;3-extracellular matrix interactions are likely to allow vascular smooth muscle cell–length autoregulation, which includes a migratory process, to maintain a narrowed lumen after a prolonged constricted state.

Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries

1 We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist. 2 Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al. 3 In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin Al inhibited the contractile response to endothelin‐1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin‐1 (100 nM) in the absence of extracellular calcium. 4 In rat α‐toxin permeabilized mesenteric arteries endothelin‐1 caused a concentration‐dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin Al did not affect endothelin‐1‐induced calcium sensitization of force. 5 We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin‐1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.

Inhibitors of Actin Filament Polymerisation Attenuate Force but Not Global Intracellular Calcium in Isolated Pressurised Resistance Arteries

Receptor-coupled contractile activation of arterial smooth muscle involves increases in intracellular calcium ([Ca2+]i) and subsequent alteration of myosin light chain phosphorylation. An additional mechanism whereby agonists could regulate vascular contractility may be alteration of actin filament dynamics. Therefore, in this study, we have investigated the influence of two inhibitors of actin filament polymerisation, cytochalasin D and latrunculin B, on the [Ca2+]i and force responsiveness of pressurised rat mesenteric arteries to α-adrenergic stimulation. Following cytochalasin D or latrunculin B treatment, phenylephrine-induced constrictions were significantly reduced to 11 ± 3.2% (n = 6) and 10 ± 4.4% (n = 6) of control, respectively, whereas [Ca2+]i remained at 98 ± 21% and 104 ± 7.0% of control, respectively. Such effects of cytochalasin D were not restricted to mesenteric small arteries. Cytochalasin D also significantly reduced the force, but not [Ca2+]i responses to agonist stimulation in other vascular (portal vein) and non-vascular (uterine) tissues. These data indicate that inhibitors of net actin polymerisation attenuate maximum agonist-induced force responsiveness without similar reductions in [Ca2+]i in pressurised resistance vessels and other smooth muscle tissues. This suggests that modulation of the dynamic equilibrium between filamentous F-actin and monomeric globular actin (G-actin) may be an important mechanism, acting independently of global [Ca2+]i homeostasis, to regulate the smooth muscle contractile state.

Comparison of U46619-, endothelin-1- or phenylephrine-induced changes in cellular Ca2+ profiles and Ca2+ sensitisation of constriction of pressurised rat resistance arteries.

In pressurised rat mesenteric small arteries (50 mmHg), we examined the effects of stimulation with U46619, endothelin‐1 (ET‐1) or phenylephrine (PE) on changes in vessel diameter, global [Ca2+]i, individual smooth muscle cell [Ca2+]i and Ca2+‐sensitisation of contraction. U46619 or ET‐1 gave tonic diameter reductions, whereas PE‐stimulated vessels gave tonic contractions or initial vasoconstrictions followed by diameter oscillations. Global [Ca2+]i changes were transient for each agonist, with tonic constrictions being accompanied by maintained submaximal global [Ca2+]i levels. U46619, ET‐1 or PE tonic constrictions were accompanied by apparently asynchronous [Ca2+]i waves in individual smooth muscle cells of the vessel wall, as examined by confocal fluorescent microscopy. In vessels exhibiting vasomotion to PE, some apparent synchrony of activation of individual cells was evident; however, this was incomplete with many cells responding out of phase with their neighbours. In α‐toxin‐permeabilised preparations, agonist‐induced Ca2+‐sensitisation of constriction at submaximal Ca2+ (pCa6.7) in the presence of GTP was greater with U46619 or ET than PE. We conclude that, in pressurised mesenteric arteries, (i) a general feature of receptor‐coupled constriction is the generation of periodic smooth muscle [Ca2+]i waves; (ii) complete synchrony of Ca2+ oscillations between smooth muscle cells is not a prerequisite for receptor‐coupled vasomotion; (iii) varied Ca2+‐sensitising actions of agonists may partly determine tonic or phasic vessel responses to different stimuli.

Middle cerebral artery structure and distensibility during developing and established phases of hypertension in the spontaneously hypertensive rat.

Objective The aims of the current study were to examine the structural properties of middle cerebral arteries (MCA) from young (5–7 weeks) and adult (20–24 weeks) spontaneously hypertensive rats (SHR), compared with age-matched Wistar–Kyoto (WKY) control rats. Design MCA segments (8–10 per group) were secured onto glass pipettes in a small vessel chamber and studied using a pressure arteriograph system. Vessels were perfused in Ca2+-free physiological salt solution to ensure the absence of tone. The wall thickness and lumen diameter were recorded at intraluminal pressures ranging from 3 to 180 mmHg using a video dimension analyser. Results There was a borderline increase in systolic pressure of the young SHR, compared with WKY controls, but the systolic pressure of the older SHR was significantly raised. The MCA lumen diameter from young SHR was reduced across the entire pressure range and arterial distensibility was not reduced, compared with WKY vessels. The MCA lumen diameter from adult SHR was reduced at high pressure, but converged with the lumen diameter of the WKY vessels at 3 mmHg, and the stress–strain relation was shifted to the left, compared with the WKY vessels; nevertheless, the slope of the tangential elastic modulus–stress relation was not significantly increased. The pressure–wall cross-sectional area relationship did not differ between strains at either time point. Conclusions These data demonstrate eutrophic inward remodelling of the MCA from young SHR, compared with WKY controls. In the adult SHR the structural changes are probably a consequence of a reduced arterial distensibility.

Acute effects of oestrogen receptor subtype-specific agonists on vascular contractility

This study shows for the first time that both the putatively selective oestrogen receptor α and oestrogen receptor β agonists PPT (4,4′,4″‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl) tris‐phenol) and DPN (2,3‐bis(4‐hydroxyphenyl)‐propionitrile) can acutely relax precontracted isolated rat mesenteric arteries at pharmacological (i.e. μM) concentrations. When compared to responses observed to similar concentrations of 17β‐oestrogen obtained on the same tissues, PPT had a significantly greater vasodilatory effect, while DPN had a significantly smaller effect. All responses were rapid being complete within 5 min exposure time. Thus, both PPT and DPN can acutely relax isolated mesenteric arteries with the relative potency of PPT>17β‐oestrogen>DPN.

Effects of the oestrous cycle and gender on acute vasodilatory responses of isolated pressurized rat mesenteric arteries to 17 β-oestradiol

The influence of the oestrous cycle and gender on responses of isolated pressurized mesenteric arteries to acute 17 β‐oestradiol was investigated. All vessels, pre‐contracted with 60 mM KCl or 10 μM U46619 (9,11 dideoxy‐11α, 9α‐epoxy methano‐prostaglandin), exhibited concentration‐dependent vasodilatory responses to 17 β‐oestradiol (3–30 μM). The largest responses were seen in vessels from female rats in pro‐oestrous (38.9±5.4% U46619 max and 63.1±4.0% KCl max for 30 μM oestradiol), the smallest from animals in di‐oestrous (20.1±3.7% U46619 and 50.1±4.5% KCL–both P<0.05 cf pro‐oestrous (all n=8)). Responses of vessels from male rats were similar to those from pro‐oestrous rats (41.5±9.1% U46619 (n=10) and 54.9±2.9% KCl (n =8)). All responsees were unaffected by inhibition of nitric oxide synthase (NOS). Female rats in pro‐oestrous had the highest plasma concentrations of 17 β‐oestradiol and testosterone (40.76±4.73 pg ml−1 and 0.29±0.05 ng ml−1 respectively (n=8)) while those in di‐oestrous had the lowest (15.24±3.94 pg ml−1 for oestradiol and 0.08±0.03 ng ml−1 for testosterone (n=8)). In male rats the concentration of oestrogen was 10.29±1.21 pg ml−1 (n=7) while that of testosterone was 3.15±0.36 ng ml−1 (n=7). Incubation of arteries isolated from male rats and from female rats in pro‐oestrous and di‐oestrous with testosterone (1 μM, 3 h) significantly enhanced the subsequent vasodilatory responses to acute 17 β‐oestradiol. Following incubation, the responses to 17 β‐oestradiol were similar in all groups. These observations suggest that gender and the oestrous cycle may influence the vascular responses to acute 17 β‐oestradiol administration.

Eutrophic Remodeling of Small Arteries in Type 1 Diabetes Mellitus Is Enabled by Metabolic Control A 10-Year Follow-Up Study

Type 2 diabetes mellitus profoundly changes small artery remodeling in response to hypertension. Abnormal increases of both wall thickness and lumen diameter are associated with an increased mortality. Changes to small artery structure in response to blood pressure (BP) in patients with type 1 diabetes mellitus have never been examined. In 1997, 17 patients with type 1 diabetes mellitus and 9 control subjects underwent in vitro assessment of gluteal-fat small arteries using pressure myography. Patients with BP <140/90 mm Hg (systolic BP: 119±3 mm Hg; n=12) had normal-resistance artery structure. However, patients with BP >140/90 mm Hg (systolic BP: 152±5 mm Hg; n=5) demonstrated vascular hypertrophic remodeling with a significant increase in the medial cross-sectional area and wall thickness. In 2008, 8 of the original 17 diabetic patients returned for a repeat assessment. All 8 of the patients had significantly improved cholesterol (2008: 154±9 mg/dL versus 1997: 191±9 mg/dL; P=0.01) and low-density lipoprotein cholesterol (2008: 79±8 mg/dL versus 1997: 122±9 mg/dL; P=0.003) but higher BPs (systolic BP: 2008: 136±3 mm Hg versus 1997: 119±6 mm Hg; P=0.03). Glycemia was improved (2008: 7.9±0.3% versus 1997: 8.9±0.6%; P=0.17), but not significantly so. In the small arteries studied, there were significant increases in medial wall thickness and wall:lumen ratio, but cross-sectional area was unchanged, indicating eutrophic remodeling. Collectively, these findings suggest that, with poor metabolic control, small arteries from patients with type 1 diabetes mellitus show hypertrophic growth in response to elevated BP, similar to that seen in type 2 diabetes mellitus. However, metabolic improvements enable eutrophic remodeling to occur in response to an increase in BP. This has only been observed previously in patients without diabetes mellitus.