Developmental and species differences in the response of the ureter to metabolic inhibition

 The effect of inhibiting oxidative phosphorylation on electrically stimulated phasic and high-K⁺ depolarization-induced tonic contractions in ureteric smooth muscle has been investigated. Intracellular [Ca²⁺] and pH were monitored fluorimetrically with simultaneous tension measurement, in adult and neonatal rat and guinea-pig ureter. Little difference was found in the response of adult or neonatal rat ureters; cyanide abolished phasic contractions and intracellular Ca²⁺ transients. The contractions of the adult guinea-pig ureter were also reduced by cyanide, but not as much as those of the adult rat. Neonatal guinea-pig was, however, remarkably resistant to the effects of cyanide, with force and Ca²⁺ transients remaining at control levels after an initial transient dip. These differences between tissues were not apparent when a high K⁺ concentration was used to depolarize tissues and produce maintained [Ca²⁺]_i and force changes; cyanide reduced force but not [Ca²⁺]_i in all preparations. Intracellular pH decreased in all preparations with inhibition of oxidative phosphorylation, but this did not correlate with changes in contraction. It is concluded that there are both species and developmental differences in the response to metabolic inhibition of the ureter which lead to differing changes in contractile activity. Received: 23 February 1998 / Received after revision: 26 March 1998 / Accepted: 27 March 1998     

Expression of inducible nitric oxide synthase in macrophages and smooth muscle cells in various types of human atherosclerotic lesions

 Nitric oxide (NO) is an important regulatory agent in blood vessels. We studied the expression of inducible nitric oxide synthase (iNOS) in different types of human atherosclerotic lesions using simultaneous in situ hybridization and immunocytochemistry. Since nitric oxide and its derivates or reaction products can have both oxidative and antioxidative effects, we also studied the presence of oxidized low-density lipoproteins (ox-LDL) and peroxynitrite-modified proteins in the same lesions as indicators of oxidative damage. Twenty-seven aortic samples were studied from seven autopsies. Samples were classified microscopically as normal areas, initial lesions (type I), fatty streaks (type II), intermediate lesions (type III), atheroma (type IV), fibroatheroma lesions (type Va) and fibrotic lesions (type Vc). In normal arterial wall iNOS mRNA was expressed at a low level in smooth muscle cells (SMCs). Absence of, or a low level of, epitopes characteristic of ox-LDL was found in the normal arterial wall. The expression of iNOS mRNA and protein was induced in macrophages and SMCs in the majority of early lesions and in all advanced atherosclerotic lesions. Epitopes characteristic of ox-LDL and peroxynitrite-modified proteins tended to be colocalized in iNOS-positive lesions. We consider that iNOS and oxidative injuries may play an important part in atherogenesis. Received: 24 November 1998 / Accepted: 2 February 1999     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

 The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells. Received: 23 May 1996 / Received after revision: 3 September 1996 / Accepted 16 September 1996     

Volume-sensitive chloride current activated by hyposmotic swelling in antral gastric myocytes of the guinea-pig

 The characteristics of volume-sensitive chloride current (I _Cl) induced by osmotic cell swelling were studied using the whole-cell patch-clamp technique and cell diameters of antral circular guinea-pig myocytes were simultaneously measured under isosmotic and hyposmotic conditions by using a video image analysis system. At –60 mV, osmotic cell swelling (200 mosmol/l) activated a sustained inward current. Instantaneous current/voltage (I-V) relations obtained by step voltage pulses showed an outward rectification. At potentials above +40 mV, the current exhibited time-dependent decay. The outward current amplitude was decreased and the reversal potential was shifted to more positive potentials by replacement of external Cl^– with gluconate^–, while the current amplitude and the I/V relation were not affected by replacing extracellular Na⁺ with N-methyl-D-glucamine. The anion permeability sequence of the swelling-induced current was I^– (1.80) > Br^– (1.31) > Cl^– (1) > F^– (0.85) > gluconate^– (0.18). The I _Cl was effectively inhibited by the Cl^– channel blockers, 4,4′-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS, 100 μM), and niflumic acid (10 μM). DIDS suppressed outward current more effectively than inward current. Also, the I _Cl was dose-dependently inhibited by arachidonic acid, an unsaturated fatty acid and also inhibited by other unsaturated fatty acids (linoleic acid and oleic acid) but not by stearic acid, a saturated fatty acid. The inhibitory effect of arachidonic acid on I _Cl was not prevented by indomethacin, a cyclo-oxygenase inhibitor and chelerythrine, a protein kinase C inhibitor. Under whole-cell patch-clamp conditions, the cell diameter was continuously measured using video image analysis, which reflects the change in cell volume. A hyposmotic-stimulation-induced increase of cell diameter was followed by I _Cl activation. In intact single gastric myocytes, relatively severe hyposmotic (176 mosmol/l) superfusing solution increased the cell diameter and the pretreatment with DIDS or with niflumic acid significantly potentiated the above effect of hyposmotic superfusion. These results suggest that volume-sensitive outwardly rectifying chloride current (I _Cl) is present in guinea-pig gastric myocyte and the I _Cl may play a role in smooth muscle cell volume regulation. Received: 6 June 1997 / Received after revision and accepted: 24 July 1997     

Fine structure of the choroidal coat of the avian eye

 To clarify further the functional anatomy of the avian choroid, including its innervation, 12 adult White-Leghorn chickens were studied by standard electron microscopy and immunoelectron microscopy with somatostatin antibody. The endothelial cells of the blood vessels in the choriocapillaris have fenestrations only facing the retina, while the nuclei are situated toward the sclera. In addition to tight junctions and zonulae adherentes, adjoining endothelial cells form gap junctions and dense plaques with attached filaments resembling those of smooth muscle cells. The fine structure of arteries and veins is similar to that of the vasculature described in other organs. The supporting tissue is organized in trabeculae, i.e., bridges of cellular and fibrous elements that surround and sustain blood and lymphatic vessels. This tissue consists primarily of a system of fusiform or star-shaped smooth muscle cells, connected to each other and to those in the vessels’ walls through macular junctions of the adherent type, less prominent than desmosomes, and perhaps also punctiform gap junctions. Occasionally, trabecular smooth muscle cells approach the lymphatic vessels, which lack a muscular tunica, and abut their endothelium with spinous appendages. This stromal muscle tissue may act as a pump for moving the lymph. The suprachoroidea consists of large lymphatic lacunae and the multilayered membrana fusca. The elongated fuscal cells form adherent junctions, tight junctions, and perhaps also gap junctions, suggesting that the membrana fusca exerts complex functions. Nerves containing myelinated axons reach the choroid and divide into smaller branches, a few of which innervate the membrana fusca. Numerous, thin nerve branches reach both the walls of arteries and veins and the trabeculae, and synaptic terminals abut the outer muscular layers of the vessel’s wall and the smooth muscle cells of the supporting tissue. Immunocytochemistry reveals the presence of numerous somatostatin-positive and somatostatin-negative axons and synaptic terminals within both trabeculae and vascular tunica media. The somatostatin-positive axons are presumed to be cholinergic axons of the choroid neurons residing in the ciliary ganglion. Taken together, these observations indicate that the avian choroid is a highly vascularized muscular sheath that may be endowed with degrees of motility and elasticity higher than those of the mammalian choroid and may therefore play an important role in compensation for experimental defocus. Accepted: 21 January 1997     

A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels

 The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca²⁺-containing (1.5 mM) solution, of 29 μm and variable lengths of 100 μm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K⁺ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged –72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba²⁺ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K⁺ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall. Received: 26 August 1997 / Received after revision and accepted: 17 October 1997     

A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes

 The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 μM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41±13 to 541±319 nM, peaked after 2–5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 μM 4-CmC, the response was small, whereas at 500 μM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 μM) or ryanodine (10 μM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems. Received: 29 October 1998 / Received after revision: 16 February 1999 / Accepted: 17 February 1999     

Light and electron microscopic examination of endothelial cells from bovine retinal vessels in long-term cultures

The aim of the present work was to examine and compare the ultrastructure of bovine retinal endothelial cells (BRECs) in vitro during several passages in a medium selective for endothelial cells. The identity of the endothelial cells was confirmed immunohistochemically, up to the tenth passage. Changes in their ultrastructure in comparison to endothelial cells in vivo occurred at the onset of culturing and not progressively with repeated passages. The cultured BRECs show high metabolic activity in all passages. While retaining their identity as endothelial cells, they modify their lipid metabolism, so that lipids are stored. This change in lipid metabolism was induced by the medium. Received: 19 May 1999 / Accepted: 4 August 1999     

Detection of skeletal muscle fatigue using an accelerometer in dynamic cardiomyoplasty

 An animal experiment was done using six mongrel dogs that weighed 28 ± 3 kg to show that an accelerometer could detect skeletal muscle fatigue in dynamic cardiomyoplasty. Through left-side thoracotomy, the heart was exposed and an electrode to sense the heartbeat was positioned on the left ventricle. A left latissimus dorsi muscle flap (LDMF) was inserted into the left chest cavity and rolled around the heart. An accelerometer was put on the rolled LDMF to sense the ventricular acceleration by contraction of the LDMF and the heart. The LDMF was stimulated under these settings: pulse width, 210 μs; stimulation output, 6 V; burst frequency, 30 Hz; burst duration, 200 ms; synchronous ratio, 1 : 4; and synchronous delay, 66 ms. Output voltage from the accelerometer was recorded 1, 3, 5, 10, and 15 min after the beginning of stimulation. Percentages of the amplitude in all dogs after 3, 5, 10, and 15 min were 81 ± 10%, 63 ± 12%, 48 ± 11%, and 45 ± 14% of the values after 1 min, respectively. Significant differences were found between the values after 1 min and those after 3 min, between the values after 3 min and those after 5 min, and between the values after 5 min and those after 10 min. This study suggests that muscle fatigue is detectable with an accelerometer in actual dynamic cardiomyoplasty. Received: May 11, 2001 / Accepted: September 10, 2002 Acknowledgments This work was financially supported in a part by a Grant in Aid for Scientific Research (05671113) from the Ministry of Education, Science, and Culture of Japan. Correspondence to:H. Kuroda     

The role of [Ca2+]i, membrane potential and pHi in the relaxation of rat mesenteric arteries to hyperosmolar acetate

 In vitro both acetate and hyperosmolarity cause vasodilation, which could be physiologically important during food ingestion and during peritoneal dialysis. The purpose of this study was to investigate the role of the intracellular calcium concentration ([Ca²⁺]_i, measured with fura-2), membrane potential (measured with glass microelectrodes) and intracellular pH [pH_i, measured with bis-carboxyethylcarboxyfluorescein (BCECF)] in the vasodilation. Hyperosmolar sodium acetate (30 mM) concentration dependently relaxed noradrenaline-precontracted arteries. This response was associated with hyperpolarization and a fall in [Ca²⁺]_i. In arteries precontracted with 50 mM K⁺ the relaxation was associated with a decrease of [Ca²⁺]_i but no change in membrane potential. Isoosmolar sodium acetate neither relaxed or affect [Ca²⁺]_i of K⁺-precontracted arteries, but induced a small relaxation with no reduction in [Ca²⁺]_i in noradrenaline-precontracted arteries. Hyperosmolar acetate caused a transient reduction of pH_i that was unrelated to relaxation. It is concluded that the mechanisms responsible for the relaxation to hyperosmolar acetate involve a decrease of [Ca²⁺]_i, which is only partly explained by hyperpolarization and probably a decrease in the sensitivity of the contractile proteins to [Ca²⁺]_i. pH_i seems not to play a role in these effects. Received: 14 January 1998 / Received after revision: 22 April 1998 / Accepted: 11 May 1998     

Capillary force-driven cell perfusion microchamber: calcium transients in isolated smooth muscle cells

 We present a new design for a sub-microlitre chamber which enables perfusion of individual cells. No equipment is required for it to be operational, as the exchange of solutions in the chamber is driven solely by capillary forces. Its active volume consists of a 30-μm-thin layer between two coverslips (separated by a couple of spacers) and is adjustable down to 0.1 μl. This slimline design (1) guarantees that all cells are kept in one (focal) plane during recording/perfusion (thus minimizing movement artefacts when intracellular fluorescence is monitored); (2) facilitates fixing of cells to a coverslip (often even without the use of poly-L-lysine, especially when small cell clusters are used); and (3) makes it possible to perfuse individual cells on a specimen stage of an upright microscope even under high-power objectives with a short working distance, which is of potential use in field studies where the smaller size of an upright microscope (compared to the inverted one) can be advantageous. As an example, we present a chamber with an active volume of approx. 0.5 μl and perfusion rate high enough to enable complete exchange of solution within 250 ms in an area of 500 μm × 500 μm. Only 1 μl of perfusing solution is required for exchanging the entire volume of the chamber. We present an example of intracellular free calcium transients in isolated smooth muscle cells upon release of intracellular sequestered calcium. Received: 13 May 1997 / Received after revision: 18 July 1997 / Accepted: 21 July 1997     

Characteristics of albumin binding to opossum kidney cells and identification of potential receptors

 Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [¹²⁵I]-labelled albumin binding at 4°C. Two binding sites were identified, one with high affinity (K _D 154.8 ±7 mg/l) and low capacity, the other with low affinity (K _D 8300 ± 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [¹²⁵I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors. Received: 24 July 1996 / Received after revision: 16 October 1996 / Accepted: 18 October 1996     

The effect of ionic strength on the kinetics of rigor development in skinned fast-twitch skeletal muscle fibres

 Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Γ/2) of the medium mainly with K⁺, imidazole⁺ and EGTA^2– to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca²⁺ (experimental temperature 18–22°C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K _m in relaxing conditions at low ionic strength. Activation by Ca²⁺ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca²⁺ induced force transients which led into a ”high rigor” state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ”high rigor” tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments. Received: 3 September 1997 / Received after revision and accepted: 12 December 1997     

Three-dimensional cytoarchitecture of rat pulmonary venous walls: a light and scanning electron microscopic study

 The three-dimensional architecture of the rat pulmonary veins was studied by light microscopy (LM) and scanning electron microscopy (SEM). For LM, the left lungs were fixed with formalin, sectioned and immunostained with an anti-α-smooth muscle actin (α-SMA) antibody in addition to conventional staining. For SEM, the specimens were fixed with glutaraldehyde and immersed in 30% KOH solution for 8 min followed by treatment of collagenase solution for more than 5 h. By LM, the smooth muscle cells stained with anti-α-SMA showed discontinuous, periodical thickenings of circular bundles in the wall of the venules, but they became thin and continuous in the larger vessels (or veins) that had a cardiac muscle layer on the outside. Under SEM, the smooth muscle cells formed circular-oriented bundles at constant intervals along the venules less than 100 μm in diameter. These bundles had circumferential constrictions in the lumen. The cardiac muscle cells, which appeared in large pulmonary veins of more than 100 μm, ran in a circular or oblique direction and completely surrounded the vessel wall outside of the thin continuous layer of smooth muscle cells. The muscle arrangements were considered to play a significant role in the return blood flow in rat pulmonary veins. Accepted: 15 June 1998     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Adaptations of the β-adrenoceptor-adenylyl cyclase system in rat skeletal muscle to endurance physical training

 β-Adrenergic mechanisms may be important in the adaptation of skeletal muscle to endurance training. β-Adrenergic signal transduction was examined in the gastrocnemius muscle of rats submitted to a progressive, 12-week treadmill running program and compared with sedentary controls. β-Adrenoceptor density was significantly lower in exercised rats than in controls. The affinity constant for [¹²⁵I]-(-) iodocyanopindolol binding was not different among the various groups. Adenosine cyclic monophosphate formation was significantly decreased in trained animals when isoproterenol plus guanosine triphosphate or forskolin plus Mn²⁺ were used to stimulate adenylyl cyclase. Immunoblot analyses revealed that the amount of the α-subunit of stimulatory guanine nucleotide-binding protein (G_s,α), both the small and the large isoforms, also decreased with physical exercise. Thus, the present report shows that endurance training results in alterations in β-adrenergic receptor density, adenylyl cyclase activity and G_s protein level in rat gastrocnemius muscle. Received: 18 October 1996 / Received after revision: 11 March 1997 / Accepted: 20 June 1997     

Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by Evans blue dye

 Whole-cell and inside-out patch-clamp techniques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolated smooth muscle cells from sheep. In whole cells Evans blue dose-dependently increased the outward current by up to fivefold. In contrast, Evans blue had no effect on inward Ca²⁺ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca²⁺-free solution, iberiotoxin (5 × 10^–8 M), or charybdotoxin (5 × 10^–8 M), but was unaffected by externally applied caffeine (5 mM) or in cells exposed to heparin (1 mg/ml) via the patch pipette. In inside-out patches bathed in a Ca²⁺ concentration of 5 × 10^–7 M, Evans blue (10^–4 M) increased the open probability of large-conductance (298-pS) Ca²⁺-dependent K⁺ channels (BK channels), shifting the half maximal-activation voltage by –70 mV. We conclude that Evans blue dye acts as an opener of BK channels. Received: 1 September 1997 / Received after revision: 18 November 1997 / Accepted: 20 November 1997