Contractile effects of endothelin-I and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K⁺ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-l-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca²⁺-free solution abolished the endothelin-l-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca²⁺-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to α-adrenoceptor blockade. Pretreatment with endothelin-1 (10^-9′ M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of ³H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [¹²⁵]endothelin-l binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.     

Contractile effects of polycations in permeabilized smooth muscle

The polycations spermine, neomycin and polylysine potentiated Ca2+-activated force in β-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in α-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by β-escin, which eliminated the contractile effect of GTPγS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca2+-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca2+-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca2+-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40μm. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR to inhibit phosphatase activity was increased by the polycations, but only at [Ca2+]<0.3μm. The results suggest that polycations increase Ca2+-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Autoradiographic localization of endothelin-1 binding sites in human colonic cancer tissue.

Endothelin-1, a potent vasoconstrictor, has been reported to stimulate mitogenesis in various types of normal and neoplastic cells and to be involved in neurotransmission. Recently, many human cancer cell lines, including those from the human colon, have been shown to produce endothelin. In this study, the occurrence of endothelin-1 binding sites was investigated in human colonic cancer tissues using in vitro autoradiography. Specific [125I]endothelin-1 binding sites were identified over tumour vessels and stromal tissues surrounding cancer cell nests. The distribution was heterogeneous, and dense silver grains were localized, especially over clusters of fibroblasts adjacent to the cancer cell nests. Endothelin binding was minimal in the cancer cells, as in the normal crypt epithelium. Quantitative analysis of the autoradiographs demonstrated high affinity (Kd = 0.50 +/- 0.06 nM; mean +/- SEM) binding sites, with a maximum binding capacity (Bmax) of 40 +/- 3.2 amol/mm2 in the cancer tissues. Our results provide evidence that specific endothelin-1 binding sites are expressed in the stromal tissues including tumour vessels, fibroblasts, and nerve fibres. Endothelin-1 may play a modulatory role in blood supply, mitogenesis, and neurotransmission in a paracrine fashion through the stromal components in human colonic cancers.     

Immunohistochemical localization of endothelin-1, endothelin-3 and endothelin receptors in human pheochromocytoma and paraganglioma

Endothelln (ET) and its receptor system have been shown to exert varlous biological effects on dlfferent types of cells In addition to their well-known vasoconstrictor activity. Recently ET-1, ET-3 and the ET₃ receptor have been shown to play an Important role In the development of neural crest-derived cells and, in this context, pheochromocytomas have been reported to harbor ET-1. Endothelin-3 or ET receptor subtypes, however, have not been examined in pheochromocytoma and paraganglioma so far. In the present study the Immunohistochemical localization of ET-1/big ET-1, ET-3/big ET-3 and the ET_A and ET_B receptors were lnvestigated to clarify the biological characteristics of these two tumors using 32 pheochromocytomas and 11 extra-adrenal paragangliomas. Endothelin-lhig ET-1 was detected in 19 pheochromocytomas (59%) and eight paragangliomas (72%), while ET-3hIg ET-3 was detected in 10 pheochromocytomas (31%) and three paragangllomas (27%). The ET_A receptor was found in 21 pheochromocytomas (66%) and In eight paragangllomas (73%), whlle the ET_B receptor was found in 25 pheochromocytomas (78%) and In eight paragangllomas (73%). Normal adrenomedullary cells lacked each antigen examined. Endothelin-immunoreactive tumor cells were dlstrlbuted focally or In a manner scattered, whlle receptor-immunostained tumor cells were distributed wlth a focal pattern for the ETa receptor and wlth a focal or diffuse pattern for the ET_B receptor. Endothelln and its receptor coexlsted In the same tumor in 21 of 28 ET-posltive pheochromocytomas and in eight of 10 ET-positlve paragangliomas. In additlon, seven pheochromocytomas and two paragangllomas revealed posltivlty of the receptor(s) irrespective of the absence of ET-immunoreactlvlty. In concluslon, ET and Its receptor are frequently and concomitantly expressed in the pheochromocytoma and paraganglloma. From the highly frequent expression of this system or the receptor(s), ET-receptor-mediated slgnal transduction of these tumors concernlng growth and/or cell survival Is expected, although definite blological slgniflcance of thls llgand-receptor system in these tumors awaits further Investigation.     

Interaction between interstitial cells and smooth muscles in the lower urinary tract and penis

Smooth muscles in the lower urinary tract and corporal tissue exhibit spontaneous contractile activity which depends on L-type Ca²⁺ channels. The mechanism underlying this activity is spontaneous electrical activity which shows varied form and property between these tissues. Recent studies revealed that interstitial cells (ICs) are widely distributed in the genitourinary system, and suggested their involvement in spontaneous muscle activity. ICs in the system are not a simple analogy of interstitial cells of Cajal (ICC) in the gut, which act as electrical pacemaker, but represent variability amongst tissues which may account for individual characteristics of each organ. In the bladder and corporal tissue, where smooth muscle cells are capable of generating spontaneous electrical activity, ICs may modulate smooth muscle activity. ICs in corporal tissue release prostaglandins via cyclooxygenase-2 (COX-2) activity and reinforce not only spontaneous but also nerve-mediated α-adrenergic contractions. In the bladder, their fundamental role in the integration of signals between populations of cells has been proposed, and thus changes in ICs may contribute to an overactive bladder, a pathological condition which results from increased excitability in detrusor smooth muscles. In the urethra, ICs may act as electrical pacemakers as do ICC. However, overall contractility of urethral smooth muscles does not necessarily rely on pacemaking of ICs, and thus some population of smooth muscles may also have their own excitability.     

Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H₂O₂ (200 μM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA₂) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H₂O₂ resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H₂O₂ treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 μM) and quercetin (50 μM). In quiesced VSMC, incubation with H₂O₂-conditioned medium resulted in increased cPLA₂ activity compared to the controls (P<.05). This activity was partially inhibited by the ET_A-receptor antagonist, PD 142893 (10 μM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H₂O₂-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.     

Contractile activation and force generation in skinned rabbit muscle fibres: effects of hydrostatic pressure.

1. Effects of hydrostatic pressure (range 0.1-10 MPa) on the isometric tension of skinned (rabbit psoas) muscle fibres were examined at 12 degrees C and at different levels of Ca2+ activation (pCa range 4-7); the effects on both the steady tension and the tension transients induced by rapid pressure release (< 1 ms) are described. 2. The steady tension was depressed by increased pressure (approximately 1% MPa-1) at a high level of Ca2+ activation (pCa approximately 4) whereas it was potentiated at lower Ca2+ levels (pCa > 6); the effects were reversible. 3. At maximal Ca2+ activation, the tension recovery following pressure release (10 MPa to atmospheric) consisted of a fast (approximately 30 s-1) and a slow (2-3 s-1) phase; the rate and the normalized amplitude (normalized to the steady tension at atmospheric pressure for a particular pCa) of the fast phase were invariant with changes in Ca2+ level. 4. The effects of changing Ca2+ level on the slow phase were complex; its positive amplitude at high Ca2+ levels changed to negative and the rate decreased to approximately 1 s-1 at low Ca2+ levels (pCa > 6.0). 5. Results are discussed in relation to previous studies on the effect of pressure on intact muscle fibres and the actin-myosin interaction. This work supports calcium regulation of cross-bridge recruitment rather than calcium regulation of the rate of a specific step in the cross-bridge cycle.     

Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells

We report for the first time that penile smooth muscle cells (SMC) not only respond to, but also synthesize, endothelin-1 (ET-1), one of the main regulators of SMC activity. Immunohistochemical studies indicated that, beside endothelial cells (EC), SMC of the human adult and fetal penis also express ET-1 and its converting enzyme, ECE-1. Accordingly, cultures of adult penile stromal cells express these genes. We also prepared and characterized penile SMC from human fetuses. These cells express SMC specific markers such as alpha smooth muscle actin and phosphodiesterase type 5A3 along with hallmarks of androgen-dependent cells (androgen receptor and 5alpha reductase type 2). Human fetal penile SMC (hfPSMC) are immunopositive for ET-1 and release ET-1. ET-1 expression in hfPSMC was strongly increased by several factors such as transforming growth factor-beta1 (TGF-beta1), interleukin-1alpha (IL-1alpha), ET-1 itself and prolonged (24 h) hypoxia. This latter condition not only affected ET-1 expression but also responsiveness. While at normal oxygen tension, hfPSMC responded to ET-1 with a decreased proliferation mediated by the endothelin-A receptors and TGF-beta1; however, during hypoxia, ET-1 stimulated cell growth. Accordingly, prolonged hypoxia up-regulated endothelin-B receptor mRNA expression. In conclusion, our results indicate that in penile tissues SMC produce ET-1 and that such production is modulated by factors involved in penile physiology and tissue remodelling. In addition, the hfPSMC we have characterized might be a useful model for studying biochemical aspects of the human erectile process in vitro.     

Neuromuscular actions of endothelin on smooth, cardiac and skeletal muscle from guinea-pig, rat and rabbit

The peptide endothelin (human, porcine) was investigated for effects on basal muscle tone and on responses to transmural nerve stimulation in a series of smooth muscle preparations, as well as in guinea-pig atrium and rat and guinea-pig diaphragm. Endothelin lacked effect on basal tone or on spontaneous and electrically driven contractions in skeletal and atrial muscle. It contracted guinea-pig ileum, pulmonary and femoral arteries, rat anococcygeus, vas deferens and urinary bladder and rabbit taenia coli, whereas guinea-pig taenia was relaxed. Guinea-pig urinary bladder and vas deferens and rabbit iris sphincter were unaffected up to 3 x 10(-8) M. Endothelin thus has a unique pattern of smooth muscle effects, exhibiting mostly contractile but also relaxing effects. Endothelin modified contractile responses to transmural nerve stimulation, yielding marked and persistent enhancement, in guinea-pig and rat vas deferens, and enhancement also in guinea-pig pulmonary artery. In guinea-pig and rat vas deferens the response to exogenous ATP was increased by endothelin, thus suggesting a strong post-junctional enhancement of neurotransmission. In guinea-pig ileum nerve-induced responses were inhibited by endothelin, whereas exogeneous acetylcholine was enhanced, an effect suggesting a simultaneous pre-junctional inhibition and post-junctional enhancement. The Ca2+ channel blocker felodipine counteracted the stimulatory effects of endothelin on tone and transmurally induced contractions. Tachyphylaxis to endothelin action was sometimes evident, but the anococcygeus being less prone to this might be useful for studies on endothelin antagonism. Endothelin thus has prominent post-junctional, and also probably pre-junctional, effects, lending further support for a distinct biological role of this peptide.     

Big endothelin-1 but not endothelin-1 is present in the smooth muscle stroma of the prostate gland of the rat

Immunohistochemical techniques were employed to localize the presence of endothelins in the mature rat prostate gland. Immunoreactivity for big endothelin‐1 but not endothelin‐1 was observed in the fibromuscular stroma of the rat prostate gland. No immunoreactivity was seen in the glandular epithelium. Double staining procedures showed big endothelin‐1 immunoreactivity to be co‐localized with α‐actin immunoreactivity. The stroma of the prostate gland also contained nerve fibres coursing through it which are immunopositive for tyrosine hydroxylase. These results suggest that big endothelin‐1 but not endothelin‐1 is co‐localized with α‐actin in the smooth muscle cells of the rat prostate gland. This implies that endothelin‐1 is synthesized on demand from big endothelin‐1 in the fibromuscular stroma of the rat prostate.     

Role of endothelin-1 in regulating proliferation of cultured human uterine smooth muscle cells

Endothelin-1 (ET-1) exhibits vasoconstricting and growth-promoting properties in vascular smooth muscle. Whether ET-1 has mitogenic properties in uterine smooth muscle cells, and which ET receptor subtype mediates this response, is unknown. The present study was undertaken to examine the proliferative potential of the ET family on human myometrial cells in culture. ET-1 stimulated DNA synthesis and proliferation of myometrial cells. The absence of a stimulating effect of endothelin-3 (ET-3) or the ETB agonist sarafotoxin 6c (S6c) was observed. The proliferative effect of 100nM ET-1 was blocked by the two ETA antagonists (BQ 123 and FR 139317), whereas the ETB antagonist IRL 1038 was ineffective. These data indicated that ET-1-induced DNA synthesis was mediated only by the ETA receptor subtype. Pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway was coupled to the ETA receptor via the Gi protein family. PTX treatment partially decreased serum-induced DNA synthesis. This suggests that some factors from serum may operate via the G- protein in initiation of mitogenesis. Insulin-like growth factors (IGFs), epidermal growth factor (EGF) and insulin were found to be mitogens in the absence of serum, and they had no potentiating effect on ET-1-induced DNA synthesis. In the presence of 0.5% serum, EGF alone caused a weak increase in DNA synthesis, while all the growth factors were able to reduce the proliferative effect of ET-1. These findings on human myometrial cells in culture raise the possibility that, under certain conditions, ET-1 may function as a positive or as a negative modulator of smooth muscle proliferation.      

Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function

Smooth muscles from the urethra and bladder display characteristic patterns of spontaneous contractile activity in the filling phase of the micturition cycle. Tonic contractions are seen in the urethral smooth muscles, and phasic contractions occur in the detrusor. Overactivity in the detrusor is a common clinical problem. The ion channels in the smooth muscle membranes play an important role in determining the functional properties, and are obvious targets for treatment of the overactive bladder. Recent evidence suggests that interstitial cells may also play a role in determining the pattern of spontaneous activity, although their precise role is less well established in the urinary tract than in the gut. The ion channels involved in these cells are also of interest. This review discusses what is known of ion channels in these tissues, and their implications for function.     

Effect of oestrogen and progesterone on the blood flow in the lower urinary tract of the rabbit

The effects of oestrogen and of oestrogen combined with progesterone were examined on the fractional distribution of cardiac output (blood flow) in the genitourinary tissues of the female rabbit. Oestradiol treatment significantly increased blood perfusion in the uterus, vagina and urethra but caused no change in the perfusion of the urinary bladder or the kidneys. The wet weight of the uterus and vagina increased significantly and in the urethra there was a tendency to weight gain following oestradiol treatment. Progesterone treatment following oestrogen appeared to reduce the effect of oestrogen on both perfusion and weight gain.     

Influence of muscle length on the force-velocity relation of K+-contractures in smooth muscle from rabbit urinary bladder

Force-velocity relations of K+-contractures of longitudinal smooth muscle from rabbit urinary bladder were studied by isotonic quick release at 37 degrees C. In order to minimize the influence of parallel elasticity the study was limited to the rising part of the length-tension curve. The force-velocity data fitted well with Hill's equation. The in situ length of the strip at a bladder volume of 10 ml is called L10. This length is 50% of that at which maximum active tension is developed. At L10 Vmax was 0.29 muscle lengths per second and it was estimated to be 0.36 lengths/s at optimum length. Constant b in Hill's equation had a value of 0.052 L10/s and it was unaffected by length changes over the interval 0.69 L10-1.44L10. At L10 a/Po was 0.17. In the interval given above, a/Po decreased with increasing length in proportion to the increase in Po, indicating that a was also length independent. According to Hill's equation [V = b(Po - P)/(P + a)], V should increase in proportion to (Po - P) when the muscle length is increased if a and b are constants. Such a linear relation was found at shorter lengths but at lengths close to or at the length for maximum active tension, V increased more than (Po - P). Two possible explanations were considered; firstly that b/(P + a) increased, and secondly that the load on the contractile element could be less that P due to an influence of the considerable tension in the parallel elastic element at these lengths. The series elastic recoil of the active muscle amounted to 3-4% of the muscle length when released to zero tension.     

Vasoactive intestinal polypeptide receptor binding sites in the human gastrointestinal tract: localization by autoradiography

Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of [125I]VIP receptor binding sites in histologically normal human surgical specimens. While the distribution of VIP binding sites was different for each gastrointestinal segment examined, specific vasoactive intestinal polypeptide binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, the circular and longitudinal smooth muscle of the muscularis externa, the myenteric plexus, and lymph nodules. In most segments, the mucosal layer expressed the highest concentration of VIP binding sites, with the duodenal and jejunal mucosa showing the highest density of receptors. These results identify putative VIP target tissues in the human gastrointestinal tract. In correlation with physiological data, VIP binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including mucosal ion transport, gastric secretion, hemodynamic regulation, gastric and intestinal motility, neuronal excitability, and modulation of the immune system.     

Effects of magnesium on45Ca uptake and release at different sites in rabbit aortic smooth muscle

Effects of 1.5 mM Mg²⁺ on muscle tension and on⁴⁵Ca uptake and release at different sites in the rabbit aortic media-intimal layer were investigated. The sustained contraction induced by either 10^?6 M norepinephrine (NE) or 60 mM K⁺ was not affected by 1.5 mM Mg²⁺ in the presence of 1.5 mM Ca²⁺. However, the contractions elicited with NE or K⁺ in 0.03 mM Ca²⁺-containing solution were inhibited by 1.5 mM Mg²⁺ by 67% and 27%, respectively. Total⁴⁵Ca uptake measured in the presence of either 1.5 mM or 0.03 mM Ca²⁺ was not affected by 1.5 mM Mg²⁺. The rate of residual⁴⁵Ca uptake (⁴⁵Ca uptake followed by a wash in La³⁺-containing solution at low temperature) measured in the presence of 1.5 mM Ca²⁺ was slightly lower in the presence of 1.5 mM Mg²⁺. However, the increase in rate of residual⁴⁵Ca uptake induced by NE or the net increase in the residual⁴⁵Ca uptake induced by K⁺ was not decreased by 1.5 mM Mg²⁺. The residual⁴⁵Ca uptake measured in the presence of 0.03 mM Ca²⁺ was reduced to 64% and 24% of controls by addition of 1.5 mM Mg²⁺ or Sr²⁺, respectively. A part of the residual⁴⁵Ca was released by NE. Uptake of⁴⁵Ca at this NE-affected Ca²⁺ site did not take place in the presence of 1.5 mM Mg²⁺ when the Ca²⁺ concentration of the medium was 0.03 mM. However, this⁴⁵Ca uptake component was only partially inhibited when the Ca²⁺ concentration of the medium was 1.5 mM. The NE-induced increase in⁴⁵Ca efflux was not inhibited by 1.5 mM Mg²⁺. From these results, Mg²⁺ appears to be a weak antagonist for both Ca²⁺ entry into the vascular smooth muscle cell and Ca²⁺ binding at a high affinity intracellular site.     

The effect of group selective reagentsN-ethylmaleimide and dithiothreitol on histamine H1-receptor binding sites in the vascular smooth muscle membranes

The chemical nature of the histamine H₁-receptors of beef aortic membranes has been elucidated by introducing two group selective reagents in the [³H]-mepyramine binding studies: dithiothreitol (DTT), a protein-disulphide group reducing reagent, andN-ethylmaleimide (NEM), a proteinthiol group alkylating agent.In the binding experiments, NEM independently inhibits [³H]-mepyramine binding. The inhibition is time and concentration dependent. DTT on the other hand potentiates the binding of the radioligand to its receptor and changes the affinity of histamine in competing for [³H]-mepyramine binding site. In the DTT-pretreated membranes (100 M), histamine shows a higher affinity for [³H]-mepyramine binding (K _i 0.35 M) than in the untreated membranes (K _i 3.7 M). Comparison of the pharmacological studies on the DTT-treated rabbit aortic strips and above binding studies, revealed a good correlation between the changes in the affinity of histamine for its receptor, when DTT was present. The results suggest an important role of the S-S and SH groups in the function of aortic histamine H₁-receptor.