Daniel J. Chaston

Non-linear relationship between hyperpolarisation and relaxation enables long distance propagation of vasodilatation

Non‐technical summary  Microvascular dilatations initiated locally in metabolically active tissues spread rapidly upstream, with little attenuation, to larger vessels whose relaxation results in the necessary increases in local blood flow. While this rapidly spreading response occurs due to the propagation of hyperpolarisation through gap junctions, it is not understood why the dilatation does not attenuate unless a regenerative electrical mechanism is involved. We show in skeletal muscle arterioles in vivo that no such regenerative electrical phenomenon exists. Instead, the local dilatation spreads without attenuation because the initial hyperpolarisation is supramaximal and the impact of voltage on dilatation is restricted to a narrow voltage window near the resting membrane potential. Knowledge of this mechanism increases our understanding of the processes which control blood flow to organs and explains how these processes can be compromised in diseases in which endothelial function is reduced.

Altered gap junctional communication and renal haemodynamics in Zucker fatty rat model of type 2 diabetes

Aims/hypothesisWe examined the link between altered gap junctional communication and renal haemodynamic abnormalities in diabetes in studies performed on Zucker lean (ZL) and the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes.MethodsThe abundance of connexin (Cx) 37, 40 and 43 was assessed by western blot and immunohistochemistry. Renal haemodynamics was characterised with GAP peptides, which are Cx mimetics, to inhibit gap junctions as a probe in both strains.ResultsZDF rats exhibited higher plasma glucose, 8-epi-prostaglandin F2α excretion, renal plasma flow and GFR than ZL rats. In ZDF rat kidney phosphorylation of Cx43 was enhanced compared with that in ZL rats. Immunohistochemical study revealed that the density of abundance of Cx37 in renin-secreting cells was significantly reduced in ZDF rats. Although renal autoregulation was markedly impaired in ZDF rats, it was preserved in ZL rats. GAP27 for Cx37,43 and for Cx40 impaired renal autoregulation in ZL rats, but failed to induce further alterations in renal autoregulation in ZDF rats.Conclusions/interpretationOur findings indicate that ZDF rats have glomerular hyperfiltration with impaired autoregulation. They also demonstrate enhanced phosphorylation of Cxs and reduced production of Cxs in ZDF rat kidney, especially of Cx37 in renin-secreting cells. Finally, our data suggest that an impairment of gap junctional communication in juxtaglomerular apparatus plays a role in altered renal autoregulation in diabetes.

Enalapril treatment alters the contribution of epoxyeicosatrienoic acids but not gap junctions to endothelium-derived hyperpolarizing factor activity in mesenteric arteries of spontaneously hypertensive rats

Reduction in endothelium-derived hyperpolarizing factor (EDHF)-mediated dilatory function in large, elastic arteries during hypertension is reversed after blood pressure normalization. We investigated whether similar mechanisms occurred in smaller mesenteric resistance arteries from aged Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs), and SHRs treated with the angiotensin-converting enzyme inhibitor, enalapril, using immunohistochemistry, serial-section electron microscopy, electrophysiology and wire myography. Unlike the superior mesenteric artery, EDHF relaxations in muscular mesenteric arteries were not reduced in SHRs, although morphological differences were found in the endothelium and smooth muscle. In WKY rats, SHRs and enalapril-treated SHRs, relaxations were mediated by small-, large-, and intermediate-conductance calcium-activated potassium channels, which were distributed in the endothelium, smooth muscle, and both layers, respectively. However, only WKY hyperpolarizations and relaxations were sensitive to gap junction blockers, and these arteries expressed more endothelial and myoendothelial gap junctions than arteries from SHRs. Responses in WKY rats, but not SHRs, were also reduced by inhibitors of epoxyeicosatrienoic acids (EETs), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and miconazole, although sensitivity to EET regioisomers was endothelium-independent in all rats. Enalapril treatment of SHRs reduced blood pressure and restored sensitivity to 14,15-EEZE, but not to gap junction blockers, and failed to reverse the morphological changes. In conclusion, the mechanisms underlying EDHF in muscular mesenteric arteries differ between WKY rats and SHRs, with gap junctions and EETs involved only in WKY rats. However, reduction of blood pressure in SHRs with enalapril restored a role for EETs, but not gap junctions, without reversing morphological changes, suggesting a differential control of chemical and structural alterations.

Chronic deficit in nitric oxide elicits oxidative stress and augments T-type calcium-channel contribution to vascular tone of rodent arteries and arterioles

AIMS As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo. METHODS AND RESULTS We used pressurized cerebral and mesenteric arteries in vitro and skeletal muscle arterioles in vivo to study the contribution of L-type (1 µmol/L nifedipine) and T-type (1 µmol/L mibefradil, 3 µmol/L NNC 55-0396) calcium channels to vascular tone, following acute or chronic inhibition of nitric oxide. Acute inhibition with l-NAME (10 µmol/L) significantly increased the T-type, but not the L-type, channel contribution to vascular tone in vitro and in vivo, and altered the smooth muscle expression of the Cav3.1 and Cav3.2 T-type channels. In pressurized mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with l-NAME, the contribution of T-type channels relative to L-type channels was significantly reduced, compared with arteries from wild-type mice.Chronic l-NAME treatment (40 mg/kg/day; 14-18 days) increased blood pressure, vascular superoxide, and the contribution of T-type channels to vascular tone in vivo. The latter was reversed by acute scavenging of superoxide with tempol (1 mmol/L), or inhibition of NADPH oxidase with apocynin (500 µmol/L) or DPI (5 µmol/L). CONCLUSION We conclude that nitric oxide deficit produces a significant increase in the contribution of Cav3.1 and Cav3.2 T-type calcium channels to vascular tone, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function.

Spreading vasodilatation in the murine microcirculation: attenuation by oxidative stress-induced change in electromechanical coupling

•  Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to upstream supply arteries. •  This is mediated by endothelial conduction of hyperpolarization, attenuation of which is expected to occur through current restriction or loss at sites of cell coupling and open ion channels in cell membranes. •  In an animal model of hypertension, we found that hyperpolarization decays more rapidly when endothelial cell coupling is reduced; however, this could not fully explain the observed attenuation in conducted vasodilatation. •  We found that increased oxidative stress, due to upregulation of angiotensin II, changed the contribution of L‐ and T‐type calcium channels to resting vessel tone. Inhibition of oxidative stress reversed this change and improved conducted vasodilatation. •  Our data suggest that cardiovascular disease may impair the ability of microvascular networks to maintain tissue integrity, due to oxidative stress‐induced changes in the way blood vessels constrict.

Polymorphism in Endothelial Connexin40 Enhances Sensitivity to Intraluminal Pressure and Increases Arterial Stiffness

Objective—To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. Approach and Results—A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. Conclusions—Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.

Loss of functional endothelial connexin40 results in exercise-induced hypertension in mice

During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacIR, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacIR protein.

260 CHRONIC IMPAIRMENT OF ENDOTHELIAL CONNEXIN40 ENHANCES ARTERIAL CONSTRICTION AND REDUCES ARTERIAL DISTENSIBILITY

Background: Synchronization of cellular activity is essential for cardiovascular function, such as the heart beat or vasocontractility. This is achieved through gap junctions and their constituent connexin (Cx) proteins. When Cx40, which highly expressed in the heart, juxtaglomerular apparatus and vascular endothelium, is globally deleted or mutated, significant cardiovascular pathology is observed in both animals and humans. However, the precise function of Cx40 in these different tissues remains unknown. We therefore aimed to determine the impact on cardiovascular function of endothelially expressed Cx40. Methods: We generated transgenic mice in which Cx40 function in the vascular endothelium was reduced through expression of a mutant form, &Dgr;Cx40, under control of the endothelium specific promoter, Tie2. We assessed active and passive properties of small mesenteric arteries by measuring the myogenic response to intraluminal pressure and determining vascular elasticity. The involvement of nitric oxide and endothelium derived hyperpolarising factor (EDHF) was determined pharmacologically, while blood pressure and renin secretion were also measured. Results: The myogenic response of mesenteric arteries from &Dgr;Cx40 mice was activated at significantly lower intraluminal pressures than in wildtype mice (EC50: Wildtype, 41.7± 0.3mmHg, n=13; &Dgr;Cx40, 33.2±0.4 mmHg, n=13; P<0.001). This occurred due to loss of contribution from EDHF. Transgenic vessels also exhibited reduced distensibility (P<0.05) without hypertrophic remodelling of the vascular wall. A modest but significant increase in blood pressure was observed in these mice without change in renin secretion. Conclusions: Life-long impairment in endothelial Cx40 enhances vasoconstriction, reduces vasodilator capacity and arterial distensibility and elevates blood pressure.

95 REDUCED NITRIC OXIDE BIOAVAILABILTY AND INCREASED REACTIVE OXYGEN SPECIES INCREASE THE CONTRIBUITON OF T-TYPE CALCIUM CHANNELS TO VASCULAR TONE

Background: Hypertension is characterised by endothelial dysfunction and reduced nitric oxide (NO) bioavailability. Approximately 30% of hypertensive patients are unresponsive to current clinical treatments. We hypothesised that this is due to changes in the calcium channels mediating vascular tone. Methods: Male C57BL/6 mice (8-12weeks) were treated with the NO inhibitor L-NAME (0.5 mg/ml) for two weeks. Blood pressure was measured by tail-cuff plethysmography. L- (1 &mgr;mol/L nifedipine) and T- (3 &mgr;mol/L NNC 55-0396) type calcium channel function was measured in cremaster muscle arterioles of anaesthetised (fentanyl 0.002, medetomidine 0.02, midazolam 0.2 mg/hr, i.v.) mice, before and after acute inhibition of reactive oxygen species (ROS) with the NADPH oxidase inhibitors, apocynin (500 &mgr;mol/L) or DPI (5 &mgr;mol/L), or the superoxide dismutase mimetic, tempol (1mmol/L). Results: Chronic L-NAME treatment significantly elevated blood pressure (120 ± 1mmHg), compared to control (104 ± 2mmHg, n = 5-6, P<0.05). Although vascular tone of cremaster muscle arterioles in vivo was not significantly altered (L-NAME: 45 ± 2%, n = 23 vs. Control: 51 ± 4% maximal diameter, n = 8, P>0.05). Chronic L-NAME treatment resulted in a significant increase in the contribution of T-type calcium channels (37 ± 2% vs. 18 ± 2%, P<0.05). Acute treatment of arterioles of L-NAME treated mice with apocynin, DPI or tempol significantly reduced vascular tone (18 ± 2%, 12 ± 6% and 24 ± 4% maximal diameter, respectively, n = 5-7, P<0.05) and the contribution of T-type calcium channels (12 ± 3%, 15 ± 4% and 7 ± 2%, respectively, P<0.05). Conclusions: T-type calcium channels contribute minimally to tone under physiological conditions in vivo, however chronic reduction in NO bioavailability, as occurs during cardiovascular disease, significantly increases T-type channel function and this is reversible by inhibition of ROS.