[Single transurethral microwave therapy of benign prostatic hyperplasia]. / Engangs transurethral mikrobølgebehandling av benign prostatahyperplasi.

We have used transurethral microwave thermotherapy in the treatment of benign prostatic hyperplasia since October 1991. Irreversible cell damage occurs when the microwaves heat the periurethral prostatic tissue. The urethra is simultaneously cooled, and is not destroyed during treatment. The patients are not hospitalized. Transurethral microwave thermotherapy is performed under local anaesthesia, no other form of analgesia has been found necessary. We have included patients with symptomatic prostatic obstruction who would otherwise have received operative or pharmacologic treatment. Patients with high residual urine, large middle lobe, urethral stricture, prostatic cancer, decreased renal function, urinary infection or metal implants were excluded. We observed a marked improvement in the Madsen symptom score after transurethral microwave thermotherapy. The score decreased from 12 before treatment to five after six weeks and 4.6 after six months (p < 0.0001). Flow increased from 8.9 to 9.7 ml/s. There was a significant reduction in the residual urine from 102 ml preoperatively to 69 ml after six months (p < 0.001). The volume of the prostate was only slightly reduced after transurethral microwave thermotherapy. Postoperative edema caused urinary retention in 13% of the patients. Two patients required transurethral resection of the prostata.

Growth-regulatory effects of glucagon, insulin, and epidermal growth factor in cultured hepatocytes. Temporal aspects and evidence for bidirectional control by cyclic AMP.

Data presented indicate that in hepatocytes insulin and glucagon promote growth by acting in a relatively early part of the prereplicative period (G0 or early G1) whereas cells (if pretreated with insulin) become more sensitive to EGF at the later stages, ie, nearer the S phase entry. The data indicate that at least two effects of glucagon (cAMP) on hepatocyte proliferation exist; in addition to a growth-promoting modulation early in the prereplicative period, there is also an inhibitory effect of glucagon (as well as other cAMP-elevating agents) that is exerted at a point shortly before the G1-to-S transition. Because both effects occur dose-dependently in the normal range of glucagon concentrations in portal blood, it is conceivable that glucagon/cAMP is involved both when liver growth is initiated and terminated.

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry.

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.

The expression of cAMP-dependent protein kinase subunits is differentially regulated during liver regeneration.

The levels of the regulatory (RI and RII) and catalytic (C) components of cAMP-dependent protein kinase and of their messages were studied during the first 36 h of liver regeneration after 70% hepatectomy. Both RI alpha mRNA and RII alpha mRNA started to increase 4 h after the resection, reaching peak levels after 9 h. RI mRNA decreased abruptly 9-12 h after resection, whereas RII mRNA stayed elevated. C alpha mRNA was rather constant during the period of study. In accordance with the mRNA data the level of C was constant while RI and RII increased during the prereplicative phase of liver regeneration. RI increased rapidly when its message became elevated. RII, however, increased noticeably only 6-8 h after its mRNA had become elevated. The increased expression of R led to a disproportion between R and C that was most pronounced 14 h after resection, i.e. coinciding with the prereplicative cAMP burst. The increased R/C ratio at that time of regeneration diminished the concentration of active C subunit during the cAMP burst. In that way the otherwise inhibitory effect of high concentrations of active C on the DNA replication may have been decreased. The fractional saturation of RI and RII by endogenous cAMP fluctuated in parallel as a function of liver cAMP levels, although there was a tendency that RI was more highly saturated than RII at high concentrations of cAMP.

DNA synthesis in cultured adult rat hepatocytes: effect of serum and calcium.

We have investigated the influence of serum and of varying Ca2+ concentrations on the DNA synthesis in primary monolayer cultures of adult rat hepatocytes, using a defined basic medium. Supplementation of the medium with 10% horse serum did not significantly affect the time course of the DNA synthesis induced by insulin and epidermal growth factor. Dose effect curves showed that serum moderately sensitized the cells to low concentrations of insulin and slightly sensitized them to epidermal growth factor, but was not required for full responses to maximal concentrations of the hormones. In the serum-free cultures a wide range of Ca2+ concentrations (0.4 - 1.8 mmol/l) yielded maximal DNA synthesis, suggesting a broad Ca2+ optimum of the S phase entry in the hepatocyte monolayers.

Effects of butyrate on epidermal growth factor receptor binding, morphology, and DNA synthesis in cultured rat hepatocytes.

We have examined the effect of butyrate on morphology, DNA synthesis, and epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. Butyrate added 2 h after plating retarded the flattening and maintained the polyhedral shape of the hepatocytes in culture. Both insulin- and EGF-stimulated DNA syntheses were slightly stimulated by butyrate at 1 mM but strongly inhibited at 5 mM. EGF receptor binding was also strongly affected by butyrate treatment of the hepatocytes. The freshly isolated hepatocytes (prior to plating) and the early-stage cultures (2 h) exhibited two classes of surface EGF receptors with high and low affinity (Kd approximately 0.05 and approximately 0.7 nM, respectively). With increasing time in culture there was a decrease in the total EGF receptor number and a corresponding reduction in the capacity for receptor-mediated EGF internalization. The high-affinity receptor class was more strongly reduced than the low-affinity class and was almost absent after 40 h in culture. Butyrate dose-dependently counteracted the decrease in the number of surface EGF receptors during culturing and preserved the high-affinity binding component. Thus, after 40 h, the cells cultured in the presence of butyrate (5 mM) had an approximately 50% elevation in the total number of receptors and the capacity to endocytose EGF compared to control cells, whereas the binding at low ligand concentration (0.02 nM) was increased 4-fold. The results suggest that butyrate, in addition to affecting morphology and DNA synthesis, also has marked effects on the hepatocyte EGF receptor status.

A simple medium for the study of hepatocyte growth in culture under defined conditions.

The combination (1:1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating.

Elevated level of beta-adrenoceptors in intact hepatocytes from partially hepatectomized rats.

The binding characteristics of 125I-iodocyanopindolol (125I-ICYP) and 3H-CGP-12177 to intact hepatocytes and a particulate fraction prepared from hepatocytes from sham-operated and partially hepatectomized rats were compared. In the particulate fraction, the beta-adrenoceptor number increased 4-10-fold after partial hepatectomy. 125I-ICYP binding to intact cells demonstrated a high fraction of non-specific binding, although phentolamine was included in the assay to reduce non-specific binding. 3H-CGP-12177 binding to intact cells also demonstrated a higher fraction of non-specific binding than in most cell types. In intact cells, the beta-adrenoceptor number increased 5-6-fold after partial hepatectomy. The number of receptors was comparable in intact cells and the broken cell preparation, indicating that receptors are conserved during the preparation of the particulate fraction.

Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis.

Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed first-order kinetics both in untreated and hormone-stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate constant for the S phase entry increased five- to sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a one-log shift of the dose-effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6-7 h vs. 26 h).

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase.

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2-acetylaminofluorene.

Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2-AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF-treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity.

Quantitative aspects of the effects of insulin, epidermal growth factor and dexamethasone on DNA synthesis in cultured adult rat hepatocytes.

Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dose-dependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24-30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nM and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 microM insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5-15 nM. When added in combination with 1.7 nM EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nM. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.

The separate estimation of cAMP intracellularly bound to the regulatory subunits of protein kinase I and II in glucagon-stimulated rat hepatocytes.

A method is described for the separate determination of cAMP intracellularly bound to the regulatory moieties (RI and RII) of protein kinase I and II. The cAMP endogenously bound to RI or RII in hepatocyte extract was adsorbed to protein A-agarose beads coated with antibodies against RI or RII. The endogenously bound cAMP was eluted from the washed beads with dilute acetic acid before being assayed. By all criteria tested, the present method did not perturb the intracellularly established equilibrium between bound and free cAMP. Stabilization of R X cAMP complexes was achieved by including sulfate in the extraction medium and sulfate/glycerol during the subsequent steps. Hepatocytes were isolated from fed male rats and contained about 0.25 pmol of RI and 0.2 pmol of RII per 10(5) cells. An intracellular titration of the cAMP binding sites of RI and RII was achieved by incubating the cells with various concentrations (1 pM to 10 nM) of glucagon. The fractional saturation of RI and RII was always similar, being 20% in nonstimulated cells. 50% saturation occurred when free cAMP was 0.46 pmol/10(5) cells. A Scatchard plot of the data for the endogenous cAMP binding suggested that cAMP interacted with RI and RII in a slightly positively cooperative manner. About 5% of the intracellularly bound cAMP was sedimentable at 10,000 X g. The apparent affinity of these particulate-associated binding sites was similar to that of soluble RI and RII. Under the conditions used no evidence was obtained for cAMP binding to other proteins than RI and RII.

A comparison of the binding characteristics of the beta-adrenoceptor antagonists 3H-dihydroalprenolol and 125I-iodocyanopindolol in rat liver.

The binding characteristics of 3H-dihydroalprenolol and 125I-iodocyanopindolol have been compared in a particulate fraction from regenerating rat liver. When total 3H-dihydroalprenolol binding and inhibition of total 3H-dihydroalprenolol binding by (-)isoprenaline, (-)alprenolol and (+/-)cyanopindolol was investigated, it was found that all agents were bound to two classes of saturable binding sites. In the inhibition studies, the presence of two binding components was not obvious until the data were transformed into Hofstee plots and these were decomposed, except in the case of (+/-)cyanopindolol. Only (+/-)cyanopindolol was found to distinguish clearly between the two saturable binding sites identified by 3H-dihydroalprenolol, as indicated by a broad plateau in the inhibition curve. When 125I-iodocyanopindolol was used as radioligand, only one saturable binding site was identified, even in the presence of less selective inhibiting ligands. The lower affinity component of 3H-dihydroalprenolol binding could be inhibited by 10 microM phentolamine. However, binding experiments with 3H-prazosin indicated that the lower affinity component was not identical with the alpha-adrenoceptor. Phentolamine did not influence 125I-iodocyanopindolol binding. Thus, due to its higher specific activity and a high degree of selectivity, 125I-iodocyanopindolol appears to be the ligand of choice.

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone.

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and beta-adrenergic receptors in cultured hepatocytes. Dependence on protein and RNA synthesis and suppression by isoproterenol.

Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes.

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.

The proliferation response of rat liver parenchymal cells after partial hepatectomy. A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine.

DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [3H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [3H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination. The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [3H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [3H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G1-fractions was only 2 and 7%, respectively.

Effect of urine pH and flow on renal clearance of methotrexate.

Hydration and urinary alkalinization are used with high doses of methotrexate (MTX) to prevent precipitation of the drug in the renal tubules and consequential nephrotoxicity. The quantitative effect of these measures on the renal clearance of MTX was studied in 8 patients with normal renal function, and in 3 patients with reduced renal function. Multiple regression analysis indicated an influence of both factors on the ratio of the renal clearances of MTX and creatinine. In the eleven patients there was a linear correlation between this ratio and urine pH (p less than 0.001); the ratio increased from 0.88 at pH 5.5 to 2.62 at pH 8.4. The pH effect on this ratio was similar in the patients with normal and reduced kidney function. An increase in urine flow did not significantly increase the ratio between renal clearance of MTX and creatinine. The effect of urinary alkalinization on renal MTX clearance could be clinically exploited in patients with delayed elimination of MTX. The probable modifying effect of alkalinization of urine on the intentionally high plasma concentration after high dose MTX infusions should be further evaluated, particularly in patients with normal renal function.