Indoxyl Sulphate Retention Is Associated with Microvascular Endothelial Dysfunction after Kidney Transplantation.

Kidney transplantation (KTx) is the preferred form of renal replacement therapy in chronic kidney disease (CKD) patients, owing to increased quality of life and reduced mortality when compared to chronic dialysis. Risk of cardiovascular disease is reduced after KTx; however, it is still a leading cause of death in this patient population. Thus, we aimed to investigate whether functional properties of the vasculature differed two years post-KTx (postKTx) compared to baseline (time of KTx). Using the EndoPAT device in 27 CKD patients undergoing living-donor KTx, we found that vessel stiffness significantly improved while endothelial function worsened postKTx vs. baseline. Furthermore, baseline serum indoxyl sulphate (IS), but not p-cresyl sulphate, was independently negatively associated with reactive hyperemia index, a marker of endothelial function, and independently positively associated with P-selectin postKTx. Finally, to better understand the functional effects of IS in vessels, we incubated human resistance arteries with IS overnight and performed wire myography experiments ex vivo. IS-incubated arteries showed reduced bradykinin-mediated endothelium-dependent relaxation compared to controls via reduced nitric oxide (NO) contribution. Endothelium-independent relaxation in response to NO donor sodium nitroprusside was similar between IS and control groups. Together, our data suggest that IS promotes worsened endothelial dysfunction postKTx, which may contribute to the sustained CVD risk.

Inappropriate heat dissipation ignites brown fat thermogenesis in mice with a mutant thyroid hormone receptor α1.

Thyroid hormone is a major regulator of thermogenesis, acting both in peripheral organs and on central autonomic pathways. Mice heterozygous for a point mutation in thyroid hormone receptor α1 display increased thermogenesis as a consequence of high sympathetic brown fat stimulation. Surprisingly, despite the hypermetabolism, their body temperature is not elevated. Here we show, using isolated tail arteries, that defective thyroid hormone receptor α1 signaling impairs acetylcholine-mediated vascular relaxation as well as phenylephrine-induced vasoconstriction. Using infrared thermography on conscious animals, we demonstrate that these defects severely interfere with appropriate peripheral heat conservation and dissipation, which in turn leads to compensatory alterations in brown fat activity. Consequently, when the vasoconstrictive defect in mice heterozygous for a point mutation in thyroid hormone receptor α1 was reversed with the selective α1-adrenergic agonist midodrine, the inappropriate heat loss over their tail surface was reduced, normalizing brown fat activity and energy expenditure. Our analyses demonstrate that thyroid hormone plays a key role in vascular heat conservation and dissipation processes, adding a unique aspect to its well-documented functions in thermoregulation. The data thus facilitate understanding of temperature hypersensitivity in patients with thyroid disorders. Moreover, the previously unrecognized connection between cardiovascular regulation and metabolic activity revealed in this study challenges the interpretation of several experimental paradigms and questions some of the currently derived hypotheses on the role of thyroid hormone in thermogenesis.

Protein kinase C activation of a blebbistatin sensitive contractile component in the wall of hypertrophying mouse urinary bladder.

AIM: To examine the role of protein kinase C (PKC) and non-muscle myosin in regulation of wall tension in the hypertrophied urinary bladder. METHODS: A partial urinary outflow obstruction was induced in the mouse. Tissue strips from sham operated controls and obstructed bladders were examined in vitro with quantitative gel electrophoresis, immunohistochemistry, and in vitro force recordings. RESULTS: Outlet obstruction (14-18 days) induced a significant growth of the bladder, 73 ± 6.13 mg compared to 19 ± 1 13 mg in sham operated controls. The hypertrophying bladder tissue had increased expression of non-muscle myosin B (SMemb) mainly localized to serosa and suburothelium. Direct activation of PKC with PDBu did not alter force in the control urinary bladder. In contrast, PDBu initiated a prominent and sustained contraction which had an increased sensitivity to the myosin type II inhibitor blebbistatin. CONCLUSIONS: PKC activates a significant contractile response in the wall of the hypertrophying urinary bladder, possibly supported by non-muscle myosin. This contractile component is not contributing to the physiological response to muscarinic stimulation, but might be separately regulated by other, yet unknown, mechanisms.

The small GTPase Rac1 is required for smooth muscle contraction.

The role of the small GTP-binding protein Rac1 in smooth muscle contraction was examined using small molecule inhibitors (EHT1864, NSC23766) and a novel smooth muscle-specific, conditional, Rac1 knockout mouse strain. EHT1864, which affects nucleotide binding and inhibits Rac1 activity, concentration-dependently inhibited the contractile responses induced by several different modes of activation (high-K+, phenylephrine, carbachol and protein kinase C activation by phorbol-12,13-dibutyrate) in several different visceral (urinary bladder, ileum) and vascular (mesenteric artery, saphenous artery, aorta) smooth muscle tissues. This contractile inhibition was associated with inhibition of the Ca2+ transient. Knockout of Rac1 (with a 50% loss of Rac1 protein) lowered active stress in the urinary bladder and the saphenous artery consistent with a role of Rac1 in facilitating smooth muscle contraction. NSC23766, which blocks interaction between Rac1 and some guanine nucleotide exchange factors, specifically inhibited the α1 receptor responses (phenylephrine) in vascular tissues and potentiated prostaglandin F2α and thromboxane (U46619) receptor responses. The latter potentiating effect occurred at lowered intracellular [Ca2+]. These results show that Rac1 activity is required for active contraction in smooth muscle, probably via enabling an adequate Ca2+ transient. At the same time, specific agonists recruit Rac1 signalling via upstream modulators, resulting in either a potentiation of contraction via Ca2+ mobilization (α1 receptor stimulation) or an attenuated contraction via inhibition of Ca2+ sensitization (prostaglandin and thromboxane receptors).

Excitation and contraction of cardiac muscle and coronary arteries of brain-dead pigs.

Excitability and contraction of cardiac muscle from brain-dead donors critically influence the success of heart transplantation. Membrane physiology, Ca2+-handling, and force production of cardiac muscle and the contractile properties of coronary arteries were studied in hearts of brain-dead pigs. Cardiac muscle and vascular function after 12 h brain death (decapitation between C2 and C3) were compared with properties of fresh tissue. In both isolated cardiomyocytes (whole-cell patch clamp) and trabecular muscle (conventional microelectrodes), action potential duration was shorter in brain dead, compared to controls. Cellular shortening and Ca2+ transients were attenuated in the brain dead, and linked to lower mRNA expression of L-type calcium channels and a slightly lower ICa,L, current, as well as to a lower expression of phospholamban. The current-voltage relationship and the current above the equilibrium potential of the inward K+ (IK1) channel were altered in the brain-dead group, associated with lower mRNA expression of the Kir2.2 channel. Delayed K+ currents were detected (IKr, IKs) and were not different between groups. The transient outward K+ current (Ito) was not observed in the pig heart. Coronary arteries exhibited increased contractility and sensitivity to the thromboxane analogue (U46619), and unaltered endothelial relaxation. In conclusion, brain death involves changes in cardiac cellular excitation which might lower contractility after transplantation. Changes in the inward rectifier K+ channel can be associated with an increased risk for arrhythmia. Increased reactivity of coronary arteries may lead to increased risk of vascular spasm, although endothelial relaxant function was well preserved.

Calprotectin is a contributor to and potential therapeutic target for vascular calcification in chronic kidney disease.

Vascular calcification is an important risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). It is also a complex process involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. In an observational, multicenter European study, including 112 patients with CKD from Spain and 171 patients on dialysis from France, we used serum proteome analysis and further validation by ELISA to identify calprotectin, a circulating damage-associated molecular pattern protein, as being independently associated with CV outcome and mortality. This was confirmed in an additional cohort of 170 patients with CKD from Sweden, where increased serum calprotectin concentrations correlated with increased vascular calcification. In primary human VSMCs and mouse aortic rings, calprotectin exacerbated calcification. Treatment with paquinimod, a calprotectin inhibitor, as well as pharmacological inhibition of the receptor for advanced glycation end products and Toll-like receptor 4 inhibited the procalcifying effect of calprotectin. Paquinimod also ameliorated calcification induced by the sera of uremic patients in primary human VSMCs. Treatment with paquinimod prevented vascular calcification in mice with chronic renal failure induced by subtotal nephrectomy and in aged apolipoprotein E-deficient mice as well. These observations identified calprotectin as a key contributor of vascular calcification, and increased circulating calprotectin was strongly and independently associated with calcification, CV outcome, and mortality in patients with CKD. Inhibition of calprotectin might therefore be a promising strategy to prevent vascular calcification in patients with CKD.

Role of Rho-kinase and protein kinase C during contraction of hypertrophic detrusor in mice with partial urinary bladder outlet obstruction.

OBJECTIVE: To study muscarinic/purinergic receptor activation and Rho-kinase/protein kinase C (PKC) signalling during smooth muscle contraction in normal and hypertrophic mouse urinary bladders. METHODS: Partial urinary outflow obstruction was induced in adult female (10-12 weeks) C57Bl/6 mice and comparisons were made with sham-operated controls. Bladder preparations were examined in vitro. Expression of signalling proteins was examined using Western blot analysis. RESULTS: Obstructed bladders increased more than threefold in weight and were found to have enhanced muscarinic and attenuated purinergic components during nerve-induced contractions. The contractile response to carbachol was shifted towards lower concentrations of carbachol for the peak response and had a markedly enhanced sustained component. The amplitude of the α,ß-methylene ATP-induced responses was lowered. Rho-kinase inhibitor Y27632 (10 µM) inhibited peak and sustained contractile responses to carbachol in control bladders (peak by 38%; plateau 57%) and obstructed bladders (peak 37% plateau 47%). PKC inhibitor GF109203X (1 µM) inhibited carbachol contractions in controls (peak by 29%; plateau 29%) and obstructed bladders (peak 17%; plateau 12%). Inhibition by a similar extent was observed after nerve stimulation. Sensitivity to Ca(2+) in high-K(+) depolarized intact tissues increased in obstructed bladders. This increased receptor-independent Ca(2+)-sensitivity was abolished by Y27632. Tissue contents of the myosin-binding phosphatase subunit MYPT-1 and catalytic phosphatase subunit PP1ß, were decreased and the contents of RhoGDI, RhoA and CPI-17 increased. A decrease in the Rho-kinase isoform ROCK-1 was observed. CONCLUSION: Based on these results, one can speculate that Rho-kinase inhibition would preferentially target the pathological phasic activity in the urinary bladder rather than inhibit the physiological receptor-mediated bladder emptying.

Unaltered size selectivity of the glomerular filtration barrier in caveolin-1 knockout mice.

The transfer of albumin from blood to tissue has been found to be increased in caveolin-1 knockout (KO) mice. This has been considered to reflect increased microvascular permeability, conceivably caused by an increased endothelial production of nitric oxide (NO) in these mice. To investigate whether such an increase in NO production would also affect glomerular barrier characteristics, the glomerular sieving coefficients (theta) to neutral FITC-Ficoll 70/400 (molecular radius 13-90 A) were determined in caveolin-1 KO mice vs. their wild-type counterparts. The theta for Ficoll were assessed using high-performance size-exclusion chromatography on blood and urine samples. Furthermore, the transcapillary escape rate (TER) of (125)I-labeled albumin and plasma volume (PV) were determined in both types of mice. The kidney expressed low levels of caveolin-1 compared with the lung and bladder, but immunofluorescence associated with vascular structures was evident. Staining was lost in the caveolin-1 KO kidney, as was caveolin-1 expression in the lung and bladder. Despite an increase in the glomerular filtration rate in caveolin-1 KO mice (0.23 +/- 0.04 vs. 0.10 +/- 0.02 ml/min; both n = 7; P < 0.05), the glomerular Ficoll sieving curves were nearly identical. Furthermore, caveolin-1 KO mice showed an increased PV (6.59 +/- 0.42 vs. 5.18 +/- 0.13 ml/100 g; P < 0.01) but only a tendency toward an increased TER (14.69 +/- 1.59 vs. 11.62 +/- 1.62%/h; not significant). It is concluded that in caveolin-1 KO mice the glomerular permeability was not increased, despite the presence of glomerular hyperfiltration. The present data are in line with the concept that the increased transvascular albumin leakage previously found in mice lacking caveolin-1 may be due to an elevation in systemic microvascular pressure due to precapillary vasodilatation, rather than being a consequence of increased microvascular permeability per se.

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.

Aortic effects of thyroid hormone in male mice.

It is well established that thyroid hormones are required for cardiovascular functions; however, the molecular mechanisms remain incompletely understood, especially the individual contributions of genomic and non-genomic signalling pathways. In this study, we dissected how thyroid hormones modulate aortic contractility. To test the immediate effects of thyroid hormones on vasocontractility, we used a wire myograph to record the contractile response of dissected mouse aortas to the adrenergic agonist phenylephrine in the presence of different doses of T3 (3,3',5-triiodothyronine). Interestingly, we observed reduced vasoconstriction under low and high T3 concentrations, indicating an inversed U-shaped curve with maximal constrictive capacity at euthyroid conditions. We then tested for possible genomic actions of thyroid hormones on vasocontractility by treating mice for 4 days with 1 mg/L thyroxine in drinking water. The study revealed that in contrast to the non-genomic actions the aortas of these animals were hyperresponsive to the contractile stimulus, an effect not observed in endogenously hyperthyroid TRß knockout mice. To identify targets of genomic thyroid hormone action, we analysed aortic gene expression by microarray, revealing several altered genes including the well-known thyroid hormone target gene hairless. Taken together, the findings demonstrate that thyroid hormones regulate aortic tone through genomic and non-genomic actions, although genomic actions seem to prevail in vivo. Moreover, we identified several novel thyroid hormone target genes that could provide a better understanding of the molecular changes occurring in the hyperthyroid aorta.

Antiphase oscillations of endothelium and smooth muscle [Ca2+]i in vasomotion of rat mesenteric small arteries.

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy. In the presence of noradrenaline and cyclopiazonic acid, ryanodine-insensitive oscillations in tone were produced. The frequency was about 1 min(-1) and amplitude about 70% of the maximal tone. The amplitude was reduced by indomethacin and increased with L-NAME. Vasomotion was inhibited by nifedipine and by 40 mM potassium. The frequency was increased and amplitude decreased by removal of the endothelium and by application of charybdotoxin and apamin. The vasomotion was associated with in-phase oscillations of membrane potential in endothelial cells and SMC and oscillations of [Ca2+]i that were in near anti-phase. We suggest a working model for the generation of oscillation based on a membrane oscillator where ion channels in both endothelial cells and SMC interact via a current running between the two cell types through myoendothelial gap junctions, which sets up a near anti-phase oscillation of [Ca2+]i in the two cell types.

Chronic cystamine treatment inhibits small artery remodelling in rats.

BACKGROUND/AIMS: We investigated whether the tissue transglutaminase inhibitor cystamine is able to inhibit remodelling of small arteries in vivo, a possibility suggested by recent in vitro experiments. METHODS: Using osmotic minipumps, phenylephrine, cystamine and/or amlodipine were infused for 1-2 weeks into 9-week-old Wistar rats. Small arteries were then removed for pressure myograph investigation. RESULTS: Phenylephrine infusion caused inward remodelling of the small arteries compared to vehicle infusion. The remodelling was abolished by concomitant infusion with cystamine; blood pressure was unaffected. Second, we investigated whether cystamine was able to inhibit outward remodelling. Rats were first infused with phenylephrine for 1 week, and some were infused for a further week with amlodipine with or without cystamine. Amlodipine caused 24% outward remodelling compared to vessels from rats at completion of the phenylephrine infusion. The outward remodelling was attenuated 86% by concomitant cystamine infusion. A series of in vitro experiments supported the inhibitory action of cystamine on tissue transglutaminase. CONCLUSION: The ability of cystamine to inhibit inward remodelling independent of blood pressure is consistent with a role of tissue transgluaminase in this process. It remains to be determined if the ability of cystamine to inhibit outward remodelling also involves inhibition of tissue transglutaminase.

NO contributes to EDHF-like responses in rat small arteries: a role for NO stores.

OBJECTIVES: Responses to EDHF are usually characterised in the presence of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors. The contribution of NO to endothelium-dependent relaxation in the presence of NOS inhibitors was assessed using NO scavengers with the objective of testing (i) whether any residual NO produces endothelium-dependent relaxation in a manner similar to EDHF and (ii) to identify the source of the residual NO. METHODS: Small rat hepatic and mesenteric arteries were mounted in a tension myograph for either isometric or membrane potential measurements. RESULTS: Relaxation to ACh was unaffected by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM), and indomethacin (Indo, 5 microM) in the absence or presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), nitro-L-arginine (300 microM) or L-nitro-mono-methyl-arginine (L-NMMA, 300 microM). Addition of OxyHb (20 microM) or carboxy-PTIO (300 microM) produced a significant suppression of ACh-induced relaxations ( approximately 40%). In L-NAME+Indo treated arteries ACh-induced hyperpolarisation (delta16.3+/-2.1 mV, n=8) was significantly suppressed with the addition of OxyHb (Delta10.2+/-1.6 mV, n=12). ACh-induced relaxation, in the presence of L-NAME+Indo+OxyHb, was abolished by raised extracellular K(+), or the combination of charybdotoxin (CTX, 100 nM)+apamin (100 nM). In contrast whilst L-NAME+indo+barium+ouabain suppressed ACh-induced relaxation, the presence of OxyHb had no additional effect. Ultraviolet light induced a relaxation in arteries treated with L-NMMA+Indo (37.0+/-5.2%, n=9) which was sensitive to OxyHb (15.2+/-10.9%, n=4), and barium+ouabain (6.39+/-2.7%, n=4), but not CTX+apamin (37.8+/-2.4%, n=4). CONCLUSIONS: These findings suggest that NO contributes significantly to the "EDHF-like" response seen in rat small arteries and that the source of this NO may be preformed vascular stores.

Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries.

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

Late onset vascular dysfunction in the R6/1 model of Huntington's disease.

Huntington's disease is a neurodegenerative disorder that also gives raise to widespread changes in peripheral organs and tissues. We tested the hypothesis that vascular dysfunction may occur in Huntington's disease by studying R6/1 mice which express exon 1 of the mutant huntingtin gene. We assessed arterial function in R6/1 and wild type (WT) mice using myography. Arterial contractility was largely unaltered in R6/1 arteries at 15 and 32 weeks of age. By 40 weeks, contractility was impaired irrespective of which vasoconstrictor we tested. Endothelium-dependent relaxation was not affected, and we observed no changes in arterial geometry or expression of contractile proteins, such as myosin regulatory light chains or smooth muscle α-actin. The frequency of calcium oscillations in R6/1 arterial smooth muscle cells was higher than in WT control tissue, whereas myosin phosphorylation was unaltered. Impairment of force by the mitochondrial inhibitors cyanide and rotenone was less pronounced in R6/1 than in WT arteries and mitochondria were enlarged, in keeping with an effect related to altered mitochondrial function. Our results reveal that arteries in the R6/1 model of Huntington's disease exhibit an age-dependent impairment of contractility and that they depend less on mitochondrial function when they contract.

AMP-activated kinase relaxes agonist induced contractions in the mouse aorta via effects on PKC signaling and inhibits NO-induced relaxation.

Adenosine monophosphate activated kinase (AMPK), a regulator of cellular metabolism, has been shown to relax arterial smooth muscle via endothelium-dependent and independent mechanisms. We have examined the role of AMPK in different smooth muscles using the activating compound, 5-amino-4-imidazolecarboxamide riboside-1-ß-d-ribofuranoside (AICAR). Isolated preparations of mouse aorta, saphenous artery, ileum and urinary bladder were compared. AICAR produced a reversible dose-dependent relaxation in aortic rings pre-incubated with AICAR and activated with phenylephrine. Less prominent relaxation was noted in the other tissues. This difference in sensitivity to AICAR was not due to differences in the expression levels of AMPK α1 mRNA. In the aorta, AICAR had a greater effect on contractions induced by phenylephrine, compared to high-K(+) induced contractions. Contractions of the aorta in response to the protein kinase C activator PDBu were prominently inhibited by AICAR. The AICAR relaxation observed in the aorta was not prevented by the NOS inhibitor L-NAME, Indomethacin or endothelium removal. Nitric oxide (NO) mediated relaxations in aortic preparations induced by acetylcholine or sodium nitroprusside (SNP) were attenuated by AICAR. In conclusion, AMPK induced relaxation of smooth muscle is tissue-dependent and most prominent in large elastic arteries. The smooth muscle relaxation is NO-independent and occurs downstream of PKC activation and is associated with attenuated relaxant responses to NO.

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.

Effects of cGMP on coordination of vascular smooth muscle cells of rat mesenteric small arteries.

OBJECTIVE: We tested the hypothesis that cGMP can induce a state of only partial coordination of vascular smooth muscle cells (VSMC). METHODS: This was done by studying the concentration-dependent effect of 8Br-cGMP on isometric and isobaric force development of noradrenaline-activated segments of rat mesenteric small arteries in which the endothelium was removed. We further measured the concentration-dependent effect of 8Br-cGMP on VSMC membrane potential, spatially resolved [Ca(2+)](i) and VSMC membrane conductance. RESULTS: With 300 microM 8Br-cGMP, coordinated [Ca(2+)](i) activity and vasomotion were seen as previously reported. At 10-30 microM 8Br-cGMP, beating isometric tension oscillations were seen. Isobaric recordings revealed oscillations with different frequencies in different parts of the arteries. At these (10-30 microM) 8Br-cGMP concentrations, membrane potential oscillations did not always concur with isometric tension oscillations, and [Ca(2+)](i) oscillations were only synchronized locally within groups of cells. 8Br-cGMP concentration-dependently decreased the frequency of vasomotion and, in unsynchronized hyperpolarized VSMC, the frequency of [Ca(2+)](i) waves. CONCLUSION: Our results demonstrated that cGMP can cause a partial coordination of the VSMC in the vascular wall (and at high concentrations near complete coordination). Furthermore, the cGMP concentration-dependent decrease of Ca(2+) wave frequency and of vasomotion frequency suggests that cGMP modifies oscillatory Ca(2+) release from the sarcoplasmic reticulum and supports the suggestion that this oscillatory release paces vasomotion.