Modulation of smooth muscle activity by nitric oxide in the human upper urinary tract

The aim of the study was to ascertain whether nitric oxide (NO) might regulate motility in the human upper urinary tract. Smooth muscle activity in the human renal pelvis and proximal ureter was studiedin vitro in organ baths, and nitric oxide synthase (NOS) activity was studied by measurement of citrulline formation. NO, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) significantly reduced the frequency of spontaneous rhythmic contractions in renal pelvis and proximal ureter. Exogenously applied NO elicited relaxations in pre-contracted renal pelvis. Calciumdependent NOS activity was significant in the renal pelvis but undetectable in the ureter. Also, NOS activity was absent in hydronephrotic renal pelvis. NO, SNP and GTN inhibited smooth muscle activity in the human upper urinary tract. NOS activity was obtained in normal renal pelvis but not in hydronephrotic renal pelvis. Regulation of urinary tract NO concentrations might offer a strategy for treatment of renal colic and disturbances in upper urinary tract motility.     

Effects of variations in extracellular pH on spontaneous contractile activity and response to nerve stimulation in smooth muscle from rat urinary bladder

The effects of variations in extracellular pH have been studied on rat detrusor muscle in vitro. At pH 7.4 a continuous low amplitude spontaneous contractile activity was found. At pH 6.75 the contractions became more regular with periods of relaxation between the contractions which had increased in amplitude. At pH 7.85 the reverse was found. The results are interpreted as a membrane effect of pH. No effect of pH on amplitudes of high-K(+)-induced contractures was found. Carbachol dose-response relations and maximal contraction amplitude to carbachol was similar at pH 7.4 and 6.75. A significant depression in response to nerve stimulation was, however, noted at pH 6.75. We suggest that, while the force output of the activated detrusor smooth muscle cell is unaffected by changes in extracellular pH, a prejunctional inhibition of nerve induced contraction might occur at low pH.     

Provisional model for propagation of electrical activity in upper urinary tract

A theory on the function of the renal pelvis as a pacemaker for ureteral peristalsis is given. The bioengineering “black box” approach is used to describe peristaltic behavior monitored at the ureter and the renal pelvis, by both peristaltic pressure and electromyographic methods. The model provides a stimulus to further thought on the nature and significance of electrical activity in the upper urinary tract.     

尿路平滑肌细胞的培养和鉴定

目的 建立尿路(膀胱、输尿管、肾盂)平滑肌细胞分离、培养、扩增的常规方法。方法人肾盂及输尿管标本各1例、人膀胱标本3例，猪输尿管标本1例、猪膀胱标本5例，采用胶原酶消化法从中分离尿路平滑肌细胞，在含10％胎牛血清的DMEM中培养，观察细胞形态及扩增情况，用免疫组化方法鉴定细胞类型特异性蛋白质α—肌动蛋白(α—actin)。结果 尿路平滑肌细胞生长良好，呈现典型的平滑肌细胞形态。细胞经6次传代仍扩增迅速，免疫组化染色显示α—actin阳性。结论 此培养方法简单、可靠，短期内可获得大量纯净的尿路平滑肌细胞。     

Prostaglandin type E activity dominates in urinary tract smooth muscle in vitro

Changes in isometric tension and spontaneous contractions in human and porcine lower urinary tract smooth muscle strips caused by PGE2, PGF2a and the prostaglandin biosynthesis inhibitor ketoprofen were investigated in vitro. Ketoprofen reduced the prostaglandin biosynthesis significantly and caused reversible and dose dependent decreases of tension and spontaneous contractions in detrusor strips, while tension increase was induced in strips from the urethra, bladder neck and trigone. PGE2 reestablished the tension and the spontaneous activity in the detrusor strips and counteracted the tension increase in strips from the urethra, bladder neck and trigone. PGF2a increased the tension in all strips. The threshold concentration for prostaglandin effect in strips pretreated with ketoprofen was 10(-10) - 10(-9) mol./l., a concentration which might be considered physiologically relevant. Prostaglandin synthesis inhibition thus caused responses opposite to those of PGE2 in every single strip. These results strongly suggest the PGE-type of activity to be more important than the PGF-type of activity in lower urinary tract smooth muscle in vitro.     

Calcium channel blockade in smooth muscle of the human upper urinary tract. I. Effects on depolarization-induced activation

The effects of the calcium blockers nifedipine, verapamil, D600 and diltiazem on mechanical activity were studied in isolated preparations of the human upper urinary tract. Two types of activity were used: spontaneous phasic-rhythmic activity in calyceal segments and potassium-induced depolarization in ureteral muscle strips. Nifedipine (10(-6) mol./l.), verapamil, D600 and diltiazem (all 10(-5) mol./l.) completely suppressed spontaneous phasic-rhythmic activity. Elevation of extracellular potassium concentration induced contractions concentration-dependently. A log-linear relationship between the extracellular calcium concentration and the 85 mmol./l. potassium-induced activation was demonstrated. Concentration-response relationships of the compounds were found by activating the muscle strips with 85 mmol./l. potassium in the Tyrode solution. This activation model produced stable and reproducible contractures. The compounds antagonized depolarization-induced activations concentration-dependently, nifedipine being the drug with the lowest EC50 value; its relative potency with reference to papaverine was about 8,000 to 1. The order of potency of the other drugs was in the following sequence: D600 greater than verapamil greater than diltiazem. It is concluded that processes in the human upper urinary tract which are triggered by depolarization (action potential or high potassium concentrations) are highly sensitive to calcium channel blockers.     

Calcium channel blockade in smooth muscle of the human upper urinary tract. II. Effects on norepinephrine-induced activation

The effects of the calcium channel blockers verapamil and nifedipine on norepinephrine-induced activation were studied in different tissues of the human upper urinary tract. In usually inactive ureteral muscle strips, norepinephrine induced predominantly phasic contractions with only minimal effects on resting tension. In contrast, in isolated segments of the renal calyx and pelvis, irrespective of preexisting spontaneous phasic activity, the same agonist effected a long-lasting tonic contraction. These different types of mechanical activity induced by norepinephrine showed different sensitivities to calcium channel blockers, phasic contractions being potently suppressed while the tonic response was little affected by the drugs. This different pattern of response to norepinephrine and the different sensitivity of the responses to calcium channel blockers suggest different and separate coupling mechanisms between the receptors involved and the calcium pools responsible for initiation of contraction. The existence of different calcium pathways activating the contractile proteins in the human upper urinary tract is postulated.     

Characterization of spontaneous depolarizations in smooth muscle cells of the Guinea pig prostate

PURPOSE: We characterized the electrical events recorded in small segments of the dorsal lobe of the prostate of immature male guinea pigs and examined some mechanisms underlying their generation. MATERIAL AND METHODS: Membrane potential recordings were made in the stroma of the guinea pig prostate using conventional single microelectrode techniques. RESULTS: Three distinct, spontaneously occurring electrical events were recorded in guinea pig prostate, namely slow waves, consisting of a depolarizing transient 14 mV in amplitude with 1 to 6 nifedipine sensitive spikes superimposed, pacemaker potentials, consisting of a larger depolarization 40 mV in amplitude, and STDs 1 to 10 mV in amplitude. Only spikes on slow waves were inhibited by nifedipine. The depolarizing transient of slow waves, pacemaker potentials and STDs were abolished by cyclopiazonic acid, a blocker of the SERCA pump, and the mitochondrial uncoupler cyanide m-chlorophenyl hydrazone as well as upon exposure to Ca(2+)-free saline or the Cl(-) channel blockers niflumic acid and anthracene-9-carboxylic acid (Sigma Chemical Co., St. Louis, Missouri). Examination of the stochastic properties of STDs revealed that they were not well modeled by Poisson statistics, but rather they occurred in a clustered manner, such they may well underlie pacemaker potential generation. CONCLUSIONS: Guinea pig prostate shows STD and pacemaker potentials that arise from the release of Ca(2+) from intracellular stores and the activation of Ca(2+) activated Cl(-) channels. We speculate that the depolarizing transient of prostatic slow waves is the propagated response of pacemaker potentials evoked at sites electrically distant from the recording electrode.     

Changes in smooth muscle cell phenotype and contractile function following ischemia-reperfusion injury in the rat urinary bladder

Twenty-eight adult male Sprague-Dawley rats were divided into four groups: Group 1 received 1 hour (h) of bilateral ischemia alone. Groups 2 and 3 received 1 h ischemia followed by 1 and 4 h of reperfusion (I-R), respectively. Group 4 consisted of age-matched control rats. Bladder strips were studied using electrical field stimulation (EFS) and KCl stimulation. Maximal contractile responses were recorded and analyzed. Temporal patterns of changes in phenotypic (non-contractile and contractile) expression of bladder smooth muscle cells were investigated using electron microscopy. The mean ratio of non-contractile to contractile phenotype (nc/c) of smooth muscle cells (SMCs) in the control group was 0.169. In the ischemia alone group, the ratio was 0.991. In the 1 h I-R group, the ratio 0.865 whereas in 4 h I-R group the ratio 1.601. The contractile responses to EFS and KCl showed decreased responses in all groups. These results clearly demonstrated that the ratio of nc/c increased significantly in the ischemia group and further increased significantly in both I-R groups. The contractile responses decreased in all ischemic groups although the magnitude of the contractile changes did not correspond in the change of phenotype ratio.     

Simultaneous recording of mechanical and intracellular electrical activity in human urinary bladder smooth muscle

OBJECTIVE: To elucidate the role of the membrane potential in human detrusor smooth muscle contraction, by simultaneously recording mechanical and intracellular electrical activity in muscle strips. Materials and methods The agonists acetylcholine and carbachol were applied to induce a contraction on muscarinic receptor stimulation; to block the response, atropine was added to the bath. The Ca2+ necessary for activating the contractile machinery can be recruited via two pathways: release from intracellular stores or influx from the extracellular matrix. High potassium was applied to induce Ca2+ influx through voltage-sensitive Ca2+ channels. RESULTS: There were significant changes in the force when agonist, antagonist and high potassium was administered. However, there were significant changes in membrane potential only when KCl was applied to the bath and not with muscarinic agonist or antagonist application. Activity in the form of spike potentials did not change significantly on applying any of the test substances. CONCLUSION: The present results indicate that the Ca2+ mobilized on M3 receptor stimulation originates primarily from intracellular stores, with no systematic changes in membrane potential. Atropine only caused a relaxation in muscle previously contracted by M3-receptor agonist stimulation; it had no effect on relaxed muscle strips.     

Cajal-like cells in the human upper urinary tract

PURPOSE: Interstitial cells of Cajal (ICCs) have an important role in the regulation of gut motility as they are responsible for the slow wave activity of smooth muscle. It is still unknown if ICCs also occur in the human upper urinary tract. Since these cells express and are marked by the c-kit receptor CD117, we investigated its occurrence and distribution along the human upper urinary tract. MATERIALS AND METHODS: Tissues from 56 human ureters, spanning proximal, middle and distal ureter segments, were analyzed by indirect immunohistochemistry using the alkaline phosphatase-anti-alkaline phosphatase method and double labeling immunofluorescence on consecutive tissue sections. Several monoclonal and polyclonal antibodies to c-kit receptor were used in combination with various cell markers for histiocytic, mast cell, endothelial, epithelial, neuronal, smooth muscle and stem cell differentiation. RESULTS: The c-kit receptor was found in 3 cell types of the ureter and in round or spindle-shaped cells. Due to their antigenic profile the first one was revealed as mast cells occurring in all layers of the ureteral wall except the urothelium. In contrast, the population of spindle-shaped cells was only marked by c-kit receptor, thus, resembling ICCs. These ICC-like cells were found among the inner and outer smooth muscle layers, and in the lamina propria. They showed a slight decrease from proximal to distal ureteral segments. However, unlike intestinal ICCs their cytomorphology differed and some cells, representing the third group of c-kit receptor positive cells, were found within the urothelium. CONCLUSIONS: Our data demonstrate the presence of ICC-like cells and their ubiquitous distribution in the human ureter. The physiological importance and pathological significance of these findings must be evaluated by functional studies and investigations of certain pathological with urinary outflow disturbance conditions.     

Haemorrhage from smooth muscle tumours of the upper gastro-intestinal tract

Eight patients with haemorrhage from smooth muscle tumours of the upper gastro-intestinal tract were treated during a 10 year period from 1973 to 1982. Seven of the tumours were benign and one a malignant leiomyoblastoma. Seven tumours were sited in the stomach and there was one duodenal lesion. Endoscopy was performed in all eight cases and made the diagnosis definitively in four. In two cases the diagnosis was confirmed on barium meal and in the other two, diagnosis was eventually made at laparotomy. Two patients were shocked on admission and required emergency surgery. In one case a diameter greater than 10 cm suggested malignant potential and wide local resection was performed. In one other case with a tumour in the antrum, a distal partial gastrectomy was performed, and in the case with leiomyoblastoma a proximal gastrectomy was performed. One case was lost to follow-up. The mean follow-up in six cases free of disease with benign tumour was 2.6 years. The patient with a tumour of greater than 10 cm in diameter remains well at 18 months follow-up and the patient with a malignant leiomyoblastoma died 2 years after surgery.     

Enkephalin inhibits presynaptically the contractility of urinary tract smooth muscle

The influence of methionine-enkephalin and leucine-enkephalin on human detrusor and on pig detrusor, trigone, bladder neck and urethral smooth muscle was investigated in vitro. The enkephalins inhibited the smooth muscle contractility presynaptically. Methionine-enkephalin was about 40% more potent than leucine-enkephalin. Phentolamine-resistant contractions were less inhibited by the enkephalins than atropine-resistant contractions and contractions that were not blocked. The inhibitory nerve responses of the trigone, bladder neck and urethra were unaffected.     

Intracellular electrical activity in human urinary bladder smooth muscle: the effect of high sucrose medium

Introduction: The primary key to pharmacotherapy of bladder instability is in the excitation-contraction coupling of detrusor smooth muscle cells. To study this process, simultaneous recordings of mechanical and electrical activity are required. However, recording of mechanical activity induces movement, which may affect the quality of intracellular recordings. Materials and Methods: We therefore compared the electrical activity of human detrusor smooth muscle cells in normal Krebs' solution and in a hypertonic solution, which immobilizes the tissue, enabling us to study the effect of movement on the membrane potential. Carbachol and KCl were applied to induce contractions. Results: Sucrose in the medium made the tissue rigid and abolished its movement, while the electrical response was not affected. When compared with recordings in normal Krebs' solution, the average resting membrane potential was not altered. However, the membrane potential was more stable, with far less spike-shaped potentials. The spike-shaped potential amplitude was larger, while the duration was decreased. Conclusions: Impairing the ability of tissue movement resulted in changes in the electrophysiological properties of detrusor smooth muscle cells. The results suggest that stretch has an effect on L-type Ca2+ channels.