Holger Nilsson

Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor.

Endothelial cells in most vascular beds release a factor that hyperpolarizes the underlying smooth muscle, produces vasodilatation, and plays a fundamental role in the regulation of local blood flow and systemic blood pressure. The identity of this endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor prostacyclin, remains obscure. Herein, we demonstrate that in mesenteric resistance arteries, release of C-type natriuretic peptide (CNP) accounts for the biological activity of EDHF. Both produce identical smooth muscle hyperpolarizations that are attenuated in the presence of high [K+], the Gi G protein (Gi) inhibitor pertussis toxin, the G protein-gated inwardly rectifying K+ channel inhibitor tertiapin, and a combination of Ba2+ (inwardly rectifying K+ channel blocker) plus ouabain (Na+/K+-ATPase inhibitor). Responses to EDHF and CNP are unaffected by the natriuretic peptide receptor (NPR)-A/B antagonist HS-142-1, but mimicked by the selective NPR-C agonist, cANF4–23. EDHF-dependent relaxation is concomitant with liberation of endothelial CNP; in the presence of the myoendothelial gap-junction inhibitor 18α-glycyrrhetinic acid or after endothelial denudation, CNP release and EDHF responses are profoundly suppressed. These data demonstrate that acetylcholine-evoked release of endothelial CNP activates NPR-C on vascular smooth muscle that via a Gi coupling promotes Ba2+/ouabain-sensitive hyperpolarization. Thus, we have revealed the identity of EDHF and established a pivotal role for endothelial-derived CNP in the regulation of vascular tone and blood flow.

Investigation of Vascular Responses in Endothelial Nitric Oxide Synthase/Cyclooxygenase-1 Double-Knockout Mice Key Role for Endothelium-Derived Hyperpolarizing Factor in the Regulation of Blood Pressure in Vivo

Background—Endothelium-dependent dilatation is mediated by 3 principal vasodilators: nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). To determine the relative contribution of these factors in endothelium-dependent relaxation, we have generated mice in which the enzymes required for endothelial NO and PGI2 production, endothelial NO synthase (eNOS) and cyclooxygenase-1 (COX-1), respectively, have been disrupted (eNOS−/− and COX-1−/− mice). Methods and Results—In female mice, the absence of eNOS and COX-1 had no effect on mean arterial blood pressure (BP), whereas BP was significantly elevated in eNOS−/−/COX-1−/− males compared with wild-type controls. Additionally, endothelium-dependent relaxation remained intact in the resistance vessels of female mice and was associated with vascular smooth muscle hyperpolarization; however, these responses were profoundly suppressed in arteries of male eNOS−/−/COX-1−/− animals. Similarly, the endothelium-dependent vasodilator bradykinin produced dose-dependent hypotension in female eNOS−/−/COX-1−/− animals in vivo but had no effect on BP in male mice. Conclusions—These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whereas NO and PGI2 are the predominant mediators in male mice. Moreover, the gender-specific prevalence of EDHF appears to underlie the protection of female eNOS−/−/COX-1−/− mice against hypertension.

Vasomotion: cellular background for the oscillator and for the synchronization of smooth muscle cells

1 Vasomotion is the oscillation of vascular tone with frequencies in the range from 1 to 20 min−1 seen in most vascular beds. The oscillation originates in the vessel wall and is seen both in vivo and in vitro. 2 Recently, our ideas on the cellular mechanisms responsible for vasomotion have improved. Three different types of cellular oscillations have been suggested. One model has suggested that oscillatory release of Ca2+ from intracellular stores is important (the oscillation is based on a cytosolic oscillator). A second proposed mechanism is an oscillation originating in the sarcolemma (a membrane oscillator). A third mechanism is based on an oscillation of glycolysis (metabolic oscillator). For the two latter mechanisms, only limited experimental evidence is available. 3 To understand vasomotion, it is important to understand how the cells synchronize. For the cytosolic oscillators synchronization may occur via activation of Ca2+‐sensitive ion channels by oscillatory Ca2+ release. The ensuing membrane potential oscillation feeds back on the intracellular Ca2+ stores and causes synchronization of the Ca2+ release. While membrane oscillators in adjacent smooth muscle cells could be synchronized through the same mechanism that sets up the oscillation in the individual cells, a mechanism to synchronize the metabolic‐based oscillators has not been suggested. 4 The interpretation of the experimental observations is supported by theoretical modelling of smooth muscle cells behaviour, and the new insight into the mechanisms of vasomotion has the potential to provide tools to investigate the physiological role of vasomotion.

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

Heptanol, 18α‐glycyrrhetinic acid (18αGA) and 18β‐glycyrrhetinic acid (18βGA) 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. Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca2+‐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. Heptanol (150 μM) 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 μM did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 18βGA (30 μM) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18αGA (100 μM) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. We conclude that in mesenteric small arteries, heptanol and 18βGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18βGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18αGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

A Cyclic GMP–dependent Calcium-activated Chloride Current in Smooth-muscle Cells from Rat Mesenteric Resistance Arteries

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 μM BAY K8644, 0.4 μM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 μM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN− > Br− > I− > Cl− > acetate > F− >> aspartate, but the conductance sequence was I− > Br− > Cl− > acetate > F− > aspartate = SCN−. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.

MECHANISMS OF CA2+ SENSITIZATION OF FORCE PRODUCTION BY NORADRENALINE IN RAT MESENTERIC SMALL ARTERIES

1 Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and α‐toxin‐permeabilized rat mesenteric small arteries. 2 The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K‐PSS (125 mM K+) and NA‐K‐PSS (K‐PSS + 10 μM noradrenaline) were titrated to 30 and 75 %, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA‐K‐PSS than with K‐PSS. With K‐PSS, the proportion of MLC phosphorylated (≈30 %) was similar at 30 and 75 % of control force; with NA‐K‐PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34 %). 3 In α‐toxin‐permeabilized arteries, the force response to 1 μM Ca2+ was increased by 10 μM noradrenaline, and MLC phosphorylation was increased from 35 to 45 %. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline‐induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATPγS in the presence of the MLC kinase inhibitor ML‐9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4 The present results show that in rat mesenteric small arteries noradrenaline‐induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

Minor role for direct adrenoceptor-mediated calcium entry in rat mesenteric small arteries.

The role of membrane potential-dependent and independent regulation of the intracellular free calcium concentration ([Ca2+]i) was assessed in the mesenteric small arteries of Wistar rats. [Ca2+]i was determined by Fura-2 fluorescence. Membrane potential measurements were made using intracellular microelectrodes. Depolarization with a high-potassium solution (K-PSS) elevated [Ca2+]i and induced contraction. Further addition of 10 microM noradrenaline (NA) did not elevate [Ca2+]i further but enhanced tone. Addition of calcium channel inhibitors (felodipine or D-600) inhibited the maintained rise in [Ca2+]i with K-PSS, but NA still elevated [Ca2+]i and force to about half the previous level. Further addition of either ryanodine or thapsigargin eliminated the rise in [Ca2+]i with NA, although 10-20% of the contraction remained. Simultaneous measurements of membrane potential, [Ca2+]i, and force during cumulative additions of NA or K-PSS in the absence of inhibitors showed similar relations between membrane potential and [Ca2+]i for each means of activation. The results indicate that membrane potential and [Ca2+]i are strongly correlated in mesenteric small arteries. A small part of the [Ca2+]i increase to NA can be attributed to release from intracellular stores. Membrane potential-independent calcium channels that are directly operated by adrenoceptors appear to play a minor role in the regulation of [Ca2+]i in these vessels.

Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y1-receptor in rat mesenteric small arteries

Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)‐elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca2+]i and tension. These effects were antagonized by the selective Y1 receptor antagonist, (R)‐N2‐(diphenacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐D‐arginineamide (BIBP 3226) (apparent pKB values of 8.54±0.25 and 8.27±0.17, respectively). NPY (0.1 μM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension‐[Ca2+]i relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA2 7.87±0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 μM) induced a maximum relaxation and a return of [Ca2+]i to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin‐relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin‐relaxed arteries with a pA2 of 7.92±0.29. Electrical field stimulation (EFS) at 8–32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non‐activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y1 receptor in intact rat mesenteric small arteries, and provide little support for involvement of other postjunctional NPY receptors in the contractile responses to NPY. Neurally released NPY also seems to act through Y1 receptors, and may serve primarily as an inhibitor of vasodilatation.

Role of Intracellular Calcium for Noradrenaline-Induced Depolarization in Rat Mesenteric Small Arteries

We have investigated the effect of intracellular calcium levels for membrane potential during noradrenaline application in isolated small arteries. Rat mesenteric small arteries were mounted for isometric tension measurement. Smooth muscle membrane potentials were measured by conventional intracellular electrodes, and intracellular calcium concentration was measured using Fura-2 fluorescence. Under control conditions, noradrenaline caused contraction and depolarization from –55.5 to –29.3 mV. In intact arteries, depleting intracellular calcium stores with thapsigargin caused smooth muscle hyperpolarization and inhibited contraction to noradrenaline. In de-endothelialized vessels, thapsigargin still depleted calcium stores, but did not affect either the depolarization or contraction caused by noradrenaline. In noradrenaline-activated vessels, inhibition of calcium influx by amlodipine caused tension and calcium levels to fall to near-baseline levels, but membrane potential returned by only 55%. Treatment with a combination of thapsigargin, D-600 and BAPTA-AM inhibited the tension and calcium responses to noradrenaline, but the membrane potential response was reduced by only 34%. Acute reduction of extracellular chloride concentration caused similar, small depolarization at rest and during noradrenaline exposure. It is concluded that an elevation of intracellular calcium concentration is not essential for noradrenaline depolarization, although part of the depolarization is associated with the raised intracellular calcium level.

Vasomotion has chloride-dependency in rat mesenteric small arteries

The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (ICl(Ca)) and cGMP-dependent (ICl(Ca,cGMP)) chloride conductances are important. The effect of anion substitution and Cl− channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl−o replacement with aspartate blocked vasomotion while 36mM SCN−o (substituted for Cl−) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca2+]i disappeared with 36mM SCN−. DIDS and Zn2+ blocked ICl(Ca,cGMP) but not ICl(Ca). Vasomotion was not sensitive to DIDS and Zn2+, and DIDS and Zn2+ induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn2+ was sensitive to 36mM SCN−o. The anion substitution data indicate that Cl− is crucial for the Vm and [Ca2+]i oscillations underlying vasomotion. The Cl− channel blocker data are consistent with both CaCCs being important.