Purinergic modulation of rat urinary bladder detrusor smooth muscle

This paper reviews the use of economic evaluations in the Swedish health care system. The most important actors are defined and examples are given how economic evaluations have played a role in the decision making process. The introduction of extracorporal shock-wave lithotripsy (ESWL) is used as an example on how economic evaluation was used for recommendations to the county councils to adopt this technology. Mammography is used as an example of how the evaluation is used in the political process following an initiative in the parliament. A study of the cost-effectiveness of hypertension treatment illustrates how economic evaluations are included as part of a medical technology assessment by SBU, the Swedish Council on Technology Assessment in Health Care. The role of economic evaluations for drug reimbursement and pricing is also reviewed. The main conclusions are that economic evaluations are one of several factors influencing a decision making process that have a strong strive for consensus. It is thus difficult to make a definitive statement of the contribution of such study to the outcome of the decision making process, and there is no evidence that the evaluation was the decisive factor. However, a number of changes in the way resources are allocated in the Swedish health care system speaks for an increasing role for such studies in the future. The county councils are identified as the main target for economic evaluations, and SBU has a key role in supplying the county councils with high quality assessment of new and old technologies.     

Increased active stress generation of denervated rat intestinal smooth muscle: functional analysis of muscarinic receptor population

The effects of denervation on the active stress production by the longitudinal muscle (LM) layer of rat jejunum were examined. Extrinsic and myenteric denervation of a segment of rat jejunum was accomplished by the serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). Isolated muscle contraction experiments revealed that the LM of the jejunum taken from rats treated with BAC 15 days before developed significantly increased active stress in response to bethanechol and carbachol, but not in response to potassium chloride. No change in -log EC50 values of any of the agonists was observed in the denervated LM layer, although a significant increase in the slope of the carbachol and bethanechol concentration-response curves was observed in the denervated LM. Schild analysis of several muscarinic antagonists revealed a 3-fold increase in the apparent dissociation constant of the M2 antagonist methoctramine in BAC-treated LM. These results suggest that the increased responsiveness of the denervated LM may originate in the muscarinic receptor population of the myocytes.     

Myofibroblast-derived smooth muscle cells during remodelling of rabbit urinary bladder wall induced by partial outflow obstruction

BACKGROUND: Fibrosis of serosa, along with smooth muscle (SM) cell hypertrophy, has been shown to occur in the rabbit bladder after partial outflow obstruction. Identification of cells involved in the serosal thickening can be of primary interest to elucidate the functional changes that this organ undergoes. EXPERIMENTAL DESIGN: Cytoskeletal protein composition of cells present in the thickened serosa at different times from the onset of obstruction (7, 15, 30 and 60 days) was evaluated. This was accomplished by means of a panel of monoclonal antibodies specific for a number of differentiation markers of mesenchymal cells (vimentin, desmin, alpha-actin of SM type, nonmuscle (NM) and SM myosins), and by immunocytochemical and immunochemical techniques. RESULTS: The immunocytochemical study revealed that cells in serosal thickening follow a two-step maturation process from pre-existing vimentin-positive cells. In the first time period (7 to 15 days of obstruction), these cells predominantly achieved an immunophenotype corresponding to that of a specific myofibroblast subtype (i.e., containing vimentin, NM myosin, and SM alpha-actin). After 30 days from the onset of obstruction, the cytoskeletal protein content of serosal cells, as also revealed by Western blotting experiments, shifted towards that of fetal-type SM cells (i.e., presence of vimentin, NM myosin, SM alpha-actin, and SM myosin isoforms). Distribution of vimentin, desmin, SM alpha-actin, and SM myosin in tissue culture as well as the ultrastructure in vivo very closely resembled that of SM cells. Bromodeoxyuridine incorporation studies indicated that cells accumulated in the serosa of obstructed bladders did not derive, at least initially, from SM cells of the detrusor muscle. CONCLUSIONS: These findings are consistent with the existence of a differentiation process in which resident mesenchymal cells of bladder serosa may transform to myofibroblasts and, subsequently, in fetal-type SM cells during experimental outflow obstruction.     

The role of the N-methyl-D-aspartic acid receptor in the relaxant effect of ketamine on tracheal smooth muscle

Ketamine and magnesium (Mg2+), well known bronchodilators, have been used to treat patients with status asthmaticus. Both can block the N-methyl-D-aspartic acid (NMDA) receptor. NMDA receptors exist in the airway, and their activation seems to be linked to the release actions of sensory neuropeptides resulting in increased airway tone. We sought to determine whether ketamine relaxes the guinea pig trachea contracted by histamine by blocking the NMDA receptor. Female guinea pigs (250-400 g) were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into strips 3 mm wide and 15 mm long. The strips were mounted in a 10-mL organ bath filled with Tyrode's solution bubbled through with 95% O2/5% CO2 at 37 degrees C. Strip contractions were measured isometrically with a force displacement transducer. We then studied the effect of NMDA receptor antagonists on histamine-induced tracheal contraction. In this protocol, we examined the effect of ketamine, Mg2+, zinc (Zn2+), or MK-801 (a noncompetitive NMDA receptor blocker) on strips contracted by 10(-5) M histamine. After full contraction was attained, ketamine (0.5-1.5 mM), MgSO4 (2-8 mM), ZnCl2(0.2-0.8 mM), or MK-801 (1.5-6 x 10(-5) M) was added, and the strip tension was measured again. We also studied the effect of NMDA on the relaxation by ketamine. After full contraction by 10(-5) M histamine, 0.5-1.5 mM KET was added alone or in combination with 0.1 mM NMDA, and the strip tension was measured again. Finally, we measured the effect of MK-801 on the relaxant effect of ketamine. After full contraction by 10(-5) M histamine, 0.5-2 mM ketamine was added alone or in combination with 0.75 or 1.5 x 10(-5) M MK-801, and the strip tension was measured again. All NMDA receptor antagonists tested reversed the tracheal contraction induced by histamine in a dose-dependent manner. However, neither the agonist NMDA nor the noncompetitive receptor blocker MK-801 affected tracheal relaxation induced by ketamine. We conclude that ketamine relaxes the tracheal smooth muscle contracted by histamine through a mechanism independent of NMDA receptors. The decreased bronchomotor tone induced by ketamine is probably due to interference with a Ca2+-requiring step necessary to maintain the contraction caused by histamine. IMPLICATIONS: Stimulation of the N-methyl-D-aspartic acid (NMDA) receptor in the airway results in airway constriction. The bronchodilator ketamine blocks the NMDA receptor. However, ketamine relaxes the guinea pig trachea contracted by histamine through a mechanism independent of the NMDA receptor.     

Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A2

When human platelets are aggregated by thrombin, material is released that rapidly contracts strips of spirally cut porcine coronary artery. Prevention of the contraction by indomethacin suggested mediation by a prostaglandin. The contraction produced by aggregating platelets was unlike those produced by prostaglandins E2, F2alpha, G2, or H2, but resembled that evoked by thromboxane A2, which is formed by platelets during aggregation.     

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.     

Enhancement of rat urinary bladder tumorigenesis by lipopolysaccharide-induced inflammation

Chronic inflammation of the urinary tract is a significant risk factor for the development of urinary bladder cancer in humans. We previously demonstrated that weekly treatment with killed Escherichia coli enhanced rat urinary bladder tumorigenesis initiated by the carcinogen N-methyl-N-nitrosourea. We conducted the present study to determine whether lipopolysaccharide (LPS), a major cell wall component of E. coli, had a tumor-enhancing effect. LPS was instilled twice a week at three doses (100, 1.0, and 0.01 microgram/ml) into heterotopically transplanted rat urinary bladders which were treated with a single low dose (0.25 mg) of N-methyl-N-nitrosourea or vehicle. Rats treated with 100 micrograms/ml of LPS showed a significant increase in the incidence and number of tumors in the bladders pretreated with N-methyl-N-nitrosourea. Treatment with LPS alone did not induce tumors. The enhancing effects were associated with a marked increase in the numbers of polymorphonuclear leukocytes and an increase in the H2O2 concentration in the bladder lumen. Oxidative stress by reactive oxygen intermediates and a proliferative response of the carcinogen-exposed urothelium to the inflammatory stimulation appeared to play a significant role in tumor enhancement by LPS.     

Transforming growth factor beta1 induces IL-1 receptor antagonist production and gene expression in rat vascular smooth muscle cells

BACKGROUND: We compared long-term results of coronary artery bypass grafting between 1976 and 1988 in 176 patients 40 years old or younger with a matched control group of 176 patients 25 to 30 years older. METHODS: Mean age was 37.4 +/- 2.7 years (+/- standard deviation) in the study group and 64.2 +/- 2.9 years in the control group. Matching criteria were age, sex, left ventricular ejection fraction, number of bypass grafts, and year of operation. RESULTS: The study group had more smokers (p = 0.000) and more patients with hypercholesterolemia (p = 0.026), unstable angina (p = 0.003), and preoperative myocardial infarction (p = 0.009); fewer patients had hypertension (p = 0.000) and diabetes (p = 0.005) in this group than in the control group. The internal mammary artery was used in 31% of the study patients and in 30% of the controls. The actuarial survival rates after 5, 10, and 15 years were 92%, 86%, and 72% in the study group and 92%, 86%, and 66% in the control group (p = 0.202). Young age was a predictor of cardiac reoperation. CONCLUSIONS: Late survival is similar for young and older patients, but the reintervention rate is higher in the younger group. The absence of unstable angina, a left ventricular ejection fraction greater than 0.45, and the use of internal mammary artery grafts increase survival in all patients.     

The use of irreversible ligands to inactivate receptor subtypes: 4-DAMP mustard and muscarinic receptors in smooth muscle

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M2 receptors in smooth muscle.     

Effects of thrombin and thrombin receptor activating peptides on rat aortic vascular smooth muscle

The role of thrombin receptor activation in isolated rat aortic rings was examined. The human thrombin receptor activating peptides (TRAPs) SFLLRNPNDKYEPF (TRAP1-14), SFLLRNP (TRAP1-7) and rat TRAP1-7 (SFFLRNP) all caused concentration-related (0.1-100 microM) contractions of endothelium-rubbed rat aortic rings. Reversal of the first two amino acids in TRAP1-14 ("reverse TRAP1-14") resulted in total loss of activity. The contractions caused by the TRAPs were reduced substantially in endothelium-intact rings due to endothelium-derived relaxing factor release because the reduced contractions were reversed by N omega-nitro-L-arginine or methylene blue. Contractions were significantly but only slightly enhanced by alpha receptor blockade and were not affected by thromboxane- or endothelin-receptor blockade or by cyclooxygenase inhibition. TRAP1-7 had no effect on contractile responses to norepinephrine, serotonin, angiotensin II or endothelin-1; however, pretreatment with nifedipine or removal of extracellular Ca++ markedly inhibited the contraction. Neither human nor rat alpha-thrombin had any contractile effect on rat aortic rings. In cultured rat aortic smooth muscle cells, alpha-thrombin (EC50 = 1.9 +/- 0.7 nM), TRAP1-14 (EC50 = 30 +/- 4 microM) and TRAP1-7 (EC50 = 20 +/- 9 microM) caused concentration-dependent increases in intracellular calcium [Ca++]i, whereas reverse TRAP1-14 was ineffective. The effect of thrombin on [Ca++]i was abolished by the thrombin inhibitor MD-805, whereas the responses to TRAP were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Stimulation of phosphoinositide hydrolysis by muscarinic receptor activation in the rat olfactory bulb

The effect of muscarinic receptor activation on phosphoinositide hydrolysis in the rat olfactory bulb was investigated by determining either the inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) mass or the accumulation of [3H]inositol phosphates ([3H]InsPs). In miniprisms of rat olfactory bulb, carbachol produced an atropine-sensitive increase in Ins(1,4,5)P3 concentration. In a membrane preparation, the formation of Ins(1,4,5)P3 was stimulated by guanosine-5'-(3-O-thio) triphosphate (GTP gamma S), but not by carbachol. However, carbachol potentiated the GTP gamma S stimulation when the two agents were combined. In miniprisms prelabelled with [3H]myo-inositol, carbachol increased the accumulation of [3H]InsPs and this effect was significantly reduced by tissue treatment with either 1 microM phorbol 12-myristate 13-acetate or 1 mM dibutyryl cyclic AMP. Analysis of concentration-response curves indicated that carbachol (EC50 = 96 microM) and oxotremorine-M (EC50 = 8.2 microM) behaved like full agonists, whereas oxotremorine, BM5, arecoline and bethanechol were partial agonists. The carbachol stimulation of [3H]InsPs accumulation was counteracted with high affinity by the M1 antagonist pirenzepine (pA2 = 8.26), and less potently by the M3 antagonist para-fluorohexahydro-sila-difenidol (pA2 = 6.7) and the M2 antagonist AF-DX 116 (pA2 = 6.12). The biochemical and pharmacological properties of the muscarinic stimulation of phosphoinositide hydrolysis were compared with those displayed by the muscarinic stimulation of adenylate cyclase in the rat olfactory bulb.     

Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in rat aortic smooth muscle cells

Creation of isopenicillin N from delta-(L-alpha-aminodipyl)-L-cysteinyl-D-valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non-heme iron-containing dioxygenase. A tripeptide R-X-S motif which consists of arginine-281 and serine-283 (Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site-directed mutagenesis arginine-281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine-283 in cIPNS was also altered by site-directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the eliminatin of the mutant's substrate-binding capability similar to that of arginine-281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R-X-S motif in the functionality of cIPNS.     

Measurement of acetylcholine released from rabbit detrusor smooth muscle using HPLC with electro-chemical detection coupled with microdialysis procedure

We measured the amount of acetylcholine (ACh) released from rabbit detrusor smooth muscles induced by electrical field stimulation (EFS) using microdialysis procedure. The dialysis probe was inserted through the detrusor muscle strip and was continuously perfused with a Ringer solution containing physostigmine sulfate, at a rate of 2 microl/min. The strip was suspended in an organ bath filled with the modified Krebs-Henseleit solution and then EFS was delivered. The isometric force was recorded and monitored in each muscle preparation. The dialysates were collected every 10 min. ACh was determined by a high performance liquid chromatography with electro-chemical detection. The contraction of the muscle strip and ACh release induced by EFS were increased in a frequency and duration dependent manner. There were some differences between frequency response curves of contraction and frequency dependent ACh release. In the contractile response, the maximum contractions were observed at lower frequencies, while ACh releases reached the maximum at higher frequencies. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. The results suggest that this new method is useful to investigate the ACh release from rabbit detrusor smooth muscles, and that other neurotransmitters than ACh possibly contribute to EFS-induced contraction.     

Tissue factor pathway inhibitor inhibits aortic smooth muscle cell migration induced by tissue factor/factor VIIa complex

Tissue factor (TF), a transmembrane glycoprotein, forms a high affinity complex with factor VII/VIIa (FVIIa) and thereby initiates blood coagulation. Tissue factor pathway inhibitor (TFPI) is an endogenous protease inhibitor of TF/FVIIa-initiated coagulation. We previously reported that TF was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). In this study, we examined the contribution of FVIIa and the effect of TFPI to TF-induced cultured SMC migration. TF/FVIIa complex showed a strong migration ability, however, neither TF alone nor FVIIa induced SMC migration. TF/FVIIa treated by a serine protease inhibitor and the complex of TF and inactivated FVIIa (DEGR-FVIIa) did not stimulate SMC migration. Pretreatment with hirudin and the antibodies to alpha-thrombin and factor X had no effect on TF/FVIIa-induced SMC migration, although alpha-thrombin and factor Xa also induced SMC migration respectively. TFPI markedly inhibited TF/FVIIa-induced SMC migration in a concentration-dependent manner, but did not affect the SMC migration induced by platelet-derived growth factor (PDGF)-BB, basic fibroblast-growth factor (bFGF), or alpha-thrombin. These results indicate that the catalytic activity of TF/FVIIa complex is important on SMC migration, and TFPI can reduce SMC migration as well as thrombosis.