G-protein coupled receptor kinase 2 and 3 expression in human detrusor cultured smooth muscle cells.

The aim of this study was to investigate the expression of G-protein coupled receptor kinases (GRKs) mRNA by RT-PCR and GRKs protein by immunohistochemistry in human detrusor cultured smooth muscle. Primary cultures of human detrusor smooth muscle cells were established using the explant method from three normal bladders. The expression of each GRK, beta-adrenergic receptor and muscarinic acethylcholine receptor (mAchR) mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining was also performed using primary antibodies for GRKs. The GRK2 and GRK3 transcripts were detected by RT-PCR. The m2, m3 and m5 mAchR and beta1, beta2 and beta3 adrenergic receptor subtypes mRNA were also detected. Using immunohistochemistry, both GRK2 and GRK3 were found to be expressed in detrusor smooth muscle cells. These results demonstrated the existence of GRK2 and GRK3 and the co-expression of m2, m3 and m5 mAchR and beta1, beta2 and beta3 adrenergic receptor subtypes in detrusor smooth muscle cells. The possibility exists that these kinases play a role in the desensitization mechanism of mAchR and beta adrenergic receptors.

Expression of the ET(A) and ET(B) endothelin receptor subtype mRNA in human detrusor cultured smooth muscle cells.

The aim of this study was to determine the expression of the endothelin receptor subtype mRNAs in human detrusor cultured smooth muscle cells using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). First strand cDNA was made from human detrusor cultured smooth muscle cells total RNA and used for PCR with primers designed to amplify fragments of the ET(A) and ET(B) endothelin receptor subtype cDNA sequences. Subcloned fragments of the ET(A) and ET(B) endothelin receptor cDNAs were used to synthesize digoxigenin-labeled cRNA probes by in vitro transcription. COS-7 cells transfected with the ET(A) and ET(B) receptor cDNAs were used as positive control and to confirm the absence of cross-hybridization due to sequence homology. Both ET(A) and ET(B) receptor mRNAs were detected by RT-PCR analysis. By ISH, both ET(A) and ET(B) receptor subtype mRNAs were detected. However, ET(A) signal was much more intense than ET(B) signal. These results indicate that mRNAs for both ET(A) and ET(B) receptors are expressed in detrusor smooth muscle cells of human urinary bladder. The ET(A) receptor is the predominant detrusor ET receptor.

Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells.

The aim of this study was to investigate the expression of the muscarinic acetylcholine receptor (mAchR) subtypes mRNA in primary culture of human prostate stromal cells using the reverse transcription polymerase chain reaction (RT-PCR), RNA blotting and in situ hybridization (ISH). Using an explant method, we obtained a primary culture of prostate stromal cells from three patients with benign prostatic hypertrophy. Total RNA was extracted using the acid guanidinium method for cDNA synthesis. First-strand cDNA was then used for PCR with primers designed to amplify the fragments of each mAchR subtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands were detected; in particular m2 transcripts was strongly detected in the stromal cell culture. Each of the PCR products were subcloned into the pGEM-T plasmid vector, sequenced and random primer labeled using 32P. Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcript. However, m3 and m4 probes did not hybridize. Using in situ hybridization (ISH), m2 receptor mRNA signals were detected in several smooth muscle cells. The staining was predominantly localized to the perinuclear cytoplasm. The m3 and m4 probes did not hybridize. These results suggested that m2 receptor subtype plays a role in smooth muscle activity of the human prostate.

Bladder neck suspension using percutaneous bladder neck stabilization to the pubic bone with a bone-anchor suture fixation system: A new extraperitoneal laparoscopic approach.

A 39-year-old woman with type 1 genuine stress urinary incontinence was treated using a novel extraperitoneal laparoscopic bladder neck suspension procedure with a bone-anchor suture fixation system. Operative time and blood loss were 2.5 and 50 g, respectively. The patient showed a remarkable improvement on chain cystography and in subjective and objective well-being with no complications, even 12 months after surgery. Extraperitoneal laparoscopic bladder neck suspension using the bone-anchor suture fixation system is an easy and feasible procedure.

alpha1- and alpha2-adrenoceptors in BPH.

The dynamic obstruction of the bladder outlet secondary to benign prostatic hyperplasia (BPH), and the contractile properties of the human prostate are mediated primarily by alpha1-adrenoceptors. There are now at least three subtypes (A, B, and D) of alpha1-adrenoceptors, and recent work revealed that alpha1A-adrenoceptor and alpha1B-adrenoceptor may have a prime role for prostatic obstruction, and contraction of artery, respectively. Very recently, the presence of a low affinity alpha1-adrenoceptor for prazosin, named alpha1L, in the human BPH tissue has been determined. Because the DNA sequence of alpha1L-adrenoceptor has not yet been cloned, the alpha1L-adrenoceptor may be another form of the alpha1A-adrenoceptor, or another pharmacologically distinct alpha1-adrenoceptor which mediates the norepinephrine-induced contraction of the prostatic smooth muscle. Furthermore, the contribution of alpha1-adrenoceptors in the prostate to symptoms (not only obstructive, but irritative symptoms) which are elicited by prostatic obstruction remains to be determined.

Pharmacological and molecular biological evidence for ETA endothelin receptor subtype mediating mechanical responses in the detrusor smooth muscle of the human urinary bladder.

The aim of this study was to characterize endothelin receptor subtypes of the detrusor muscle of the human urinary bladder. The receptor subtypes mediating endothelin (ET)-1-induced activity in the human detrusor smooth muscles have been characterized using isometric contraction and reverse transcription-polymerase chain reaction (RT-PCR). ET-1 (a non-selective ET receptor agonist; 10(-10) M to 10(-6) M) exhibited concentration-dependent contractions in human urinary bladder with a plateau at concentrations above 3x10(-7) M. Neither IRL1620 nor sarafotoxin S6c (both ETB-selective agonists; 10(-10) M to 10(-6) M) elicited contractile activity in the human urinary bladder detrusor smooth muscle. FR139317 (an ETA-selective antagonist; 10(-7) M to 10(-5) M) produced a marked shift to the right of the ET-1 concentration-response curve in human urinary bladder detrusor smooth muscle (from the Schild plot TpA2=7.96; slope=0.95). In contrast, RES701-1 (an ETB-selective antagonist; 10(-7) M to 10(-5) M) had no effect on the ET-1 concentration-response curve. RT-PCR revealed positive amplification of ETA receptor mRNA fragment, but not ETB. These results indicate that the ET-1-induced contractile effects of urinary bladder detrusor smooth muscle seem to be mediated mainly by the ETA receptor, not by the ETB receptor.

Evidence for beta3-adrenoceptor subtypes in relaxation of the human urinary bladder detrusor: analysis by molecular biological and pharmacological methods.

The purpose of the present study was to confirm the presence of beta3-adrenoceptor subtype in the relaxation of human urinary bladder detrusor tissue by reverse transcription-polymerase chain reaction (PCR); direct sequencing of the PCR product, in situ hybridization; and isometric contraction. Using reverse transcription-PCR, the mRNAs of three receptor subtypes (beta1, beta2, and beta3) were expressed in the human urinary bladder detrusor tissue. Direct sequencing of the PCR product of the above beta3-adrenoceptor revealed no mutation in the amplified regions. In situ hybridization with digoxygenin-labeled oligonucleotide probe revealed the presence of the mRNA of beta3-adrenoceptor subtype in the smooth muscle of the urinary bladder. The relaxant effects of isoproterenol (a nonselective beta-adrenoceptor agonist); ZD7114, BRL37344, and CGP12177A (putative selective beta3-adrenoceptor agonists); and SR59230A (a putative selective beta3-adrenoceptor antagonist) were tested using an isometric contraction technique. Isoproterenol in either the presence or absence of both atenolol (a beta1-adrenoceptor-selective antagonist) and butoxamine (a beta2-adrenoceptor-selective antagonist) revealed a relaxant effect on the carbachol-induced contraction of the human urinary bladder detrusor. Both BRL37344 and CGP12177A also revealed relaxant effects on the human urinary bladder detrusor, but ZD7114 did not elicit any relaxation. These results suggest that beta3-adrenoceptor may have some role in urine storage in the human urinary bladder.

Alpha-1 adrenoceptor subtypes (high, low) in human benign prostatic hypertrophy tissue according to the affinities for prazosin.

BACKGROUND: A novel classification of alpha-1 adrenoceptor subtypes (High, Low) was applied to human benign prostatic hypertrophy (BPH) tissue. METHODS: Human BPH specimens were examined by a radioligand binding assay method using 3H-prazosin, and those data were compared with preoperative therapies. RESULTS: (1) Scatchard analysis showed a high-affinity site (Kd:27.18 +/- 6.41 pM; Bmax:9.29 +/- 0.98 fM/mg protein; mean +/- SE) as alpha 1H, and a low-affinity site (Kd: 4088.0 +/- 744.34 pM, Bmax: 140.81 +/- 19.98 fM/mg protein) as alpha 1L subtype, for prazosin. (2) The Kd and Bmax were not different in the nontreated group (n = 5), alpha 1 blocker group (n = 5), and antiandrogen group (n = 5), in either alpha 1-high affinity or alpha 1-low affinity subtype. (3) Phenoxybenzamine had different pKi values for the above two adrenoceptor subtypes. Scatchard analysis showed that alpha 1-high affinity binding site disappeared in the presence of 1 microM of phenoxybenzamine, and the Kd and Bmax values in the presence of 1 microM of phenoxybenzamine were almost identical to the alpha 1-low affinity site of the two subtypes. CONCLUSIONS: Human BPH tissue possesses both alpha 1H- and alpha 1L-adrenoceptor subtypes according to the affinities for prazosin, and only the alpha 1H subtype can be completely inhibited by some concentration of phenoxybenzamine. Treatment by alpha 1 blocker may not change the conditions of alpha 1-adrenoceptors in prostatic tissue.

The alpha-adrenoceptor antagonist properties of the enantiomers of doxazosin in the human prostate.

The alpha-adrenoceptor antagonist properties of doxazosin and its enantiomers were characterized using human prostate tissue and cell membranes isolated from rat-1 fibroblast expressing each of the cloned human alpha 1-adrenoceptor subtypes. In the alpha 1-adrenoceptor-binding studies on the human prostate with [3H]doxazosin and 2-{[beta-(3-[125I],4-hydroxyphenyl)ethyl]aminomethyl}-l-tetralone ([125I]HEAT), no significant differences were observed between racemic doxazosin, R-doxazosin and S-doxazosin (mean -log Ki (pKi) values were 8.60-8.63, 8.47-8.55 and 8.61-8.65, respectively), whereas the alpha 2-adrenoceptor-binding studies with [3H]rauwolscine and [3H]clonidine revealed that the alpha 2-adrenoceptor-binding affinity of S-doxazosin (pKi = 5.91-5.94) was slightly (3- or 4-fold), but significantly lower than that of R-doxazosin (pKi = 6.47-6.54). Studies in phenylephrine-contracted prostatic tissue showed no significant difference in alpha 1-adrenoceptor antagonist potency between racemic doxazosin, R-doxazosin and S-doxazosin (pA2 values were 8.43 +/- 0.28, 8.64 +/- 0.56 and 8.75 +/- 0.38, respectively). In the binding studies with cloned alpha 1-adrenoceptor subtypes using [3H]prazosin and [125I]HEAT, racemic doxazosin, R-doxazosin and S-doxazosin showed no selectivity for the alpha 1-adrenoceptor subtypes. The present study demonstrated that doxazosin and its enantiomers are highly selective alpha 1-adrenoceptor antagonists and that there is no evidence suggesting differential alpha 1-adrenoceptor antagonist effects of doxazosin and its enantiomers in the human prostate. Doxazosin, therefore, could be described as displaying balanced activity across all three alpha 1-adrenoceptor subtypes.

Genetic engineering of the processing site of D1 precursor protein of photosystem II reaction center in Chlamydomonas reinhardtii.

The D1 protein (D1) of photosystem II (PSII) reaction center is synthesized as a precursor (pD1) and then processed at its carboxyl terminus to establish the function of water cleavage. The amino acid sequence of the carboxyl terminal extension excised by this process is poorly conserved except for a residue after the cleavage site at position of 345. We have constructed a vector for site-directed mutagenesis of the chloroplast psbA gene encoding D1 of the green alga, Chlamydomonas reinhardtii. The vector enables one to transform the chloroplasts of a psbA deletion mutant (Fud7) and directly select transformants for resistance to spectinomycin. Using this transforming vector, we have substituted Ser345 to Gly, Cys, Val and Phe in order to investigate effects of the amino acid side chain at this position on the processing rate. All of the resulting transformants exhibited the PSII activity as wild type and grew normally under photoautotrophic conditions even under strong light where rapid turnover of D1 protein is expected to occur. Western blotting analysis demonstrated that mature D1 accumulates in these transformants at wild type level. Pulse and chase labeling of chloroplast-encoded proteins using [35S] sulfate revealed that the processing of D1 precursor protein occurs in all four transformants as efficiently as in wild type, at least under the experimental conditions examined. The results suggest that either the amino acid side chain at position of 345 (+1 position) is not crucial to the enzymatic cleavage of pD1 in vivo or the apparent rate of processing in vivo is not limited by the enzymatic cleavage.

Pharmacological evidence of distinct alpha 1-adrenoceptor subtypes mediating the contraction of human prostatic urethra and peripheral artery.

1. The alpha 1-adrenoceptor subtypes mediating contractions of the smooth muscle in human prostatic urethra and branches of internal iliac artery were characterized in isometric contraction experiments. 2. Phenylephrine produced concentration-dependent contractions in both the urethra and artery. These responses were competitively inhibited by prazosin, WB4101 and 5-methyl-urapidil, and the slopes of Schild plots for all these antagonists were close to unity. 3. The pA2 values for prazosin were not significantly different between the urethra (9.42 +/- 0.11; mean +/- s.d.) and artery (9.50 +/- 0.27), while the pA2 values for WB4101 and 5-methyl-urapidil in the human prostatic urethra (8.94 +/- 0.19 and 8.42 +/- 0.14, respectively) were significantly greater than in the branches of human internal iliac artery (7.94 +/- 0.21 and 7.43 +/- 0.22, respectively; P < 0.01). 4. Pretreatment with chlorethylclonidine (CEC) at concentrations ranging from 0.1 microM to 100 microM attenuated the maximum contraction to phenylephrine in a concentration-dependent manner in both the urethra and artery. However, the urethra was significantly less affected by CEC than the artery. The pD'2 values (negative logarithm of the molar concentration of antagonist which reduced the maximum contraction to one half) in the urethra and artery were 4.35 +/- 0.27 and 5.20 +/- 0.37, respectively (P < 0.01). 5. The present results indicate that there are distinct populations of alpha 1-adrenoceptor subtypes in the human prostatic urethra and branches of the internal iliac artery. The alpha 1-adrenoceptors responsible for the contraction of the human internal iliac artery branches are predominantly alpha 1 B-subtype, whereas those in the human prostatic urethra are considered to be not alpha 1 B, but alpha 1 c or possibly alpha 1 A or alpha 1A/D-subtype.

[Survey of activities of daily living in non-institutionalized elderly residents].

To investigate the states of activities of daily living (ADLs) in the elderly, non-institutionalized residents-aged 65 and over living in Azuchi (population of approximately 12,000) were interviewed. Data was collected from 1,287 subjects (509 men and 778 women; 92.1%) for the 3 years, 1990 to 1992. Differences in difficulties with the 6 basic ADLs (BADLs) and the 7 instrumental ADLs (IADLs) were analyzed according to gender and age. For the BADLs (eating, dressing, bathing, using the toilet, mobility, and incontinence), only the percentage of incontinence was significantly higher in women than in men (p < 0.01). The percentage of those fully independent in the 6 BADLs in men was 87%, which was higher than that in women (77%), but except for incontinence there were no significant differences between the sexes. The percentages of disability in the BADLs for women and for both sexes combined were higher according to age (p < 0.01), but for men, only those of dressing (p < 0.05) and of incontinence (p < 0.01) were related with age. The disability percentage for cooking was the only activity among the IADLs (cooking, using the telephone, cleaning house, walking, shopping, going up and down steps, and using transportation) which was significantly higher in men (p < 0.01), but those for walking, going up and down steps, and using transportation were higher in women (p < 0.01). The percentage of those who were fully independent was 74% in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of polychlorinated biphenyls on the growth of chicken embryos.

Incubation of chicken embryos with either 0.01 or 0.03 ppm polychlorinated biphenyls (PCBs) for 12, 15, or 18 d resulted in a significant decrease in liver and body weight associated with enhanced mortality. Teratological examination revealed an increased frequency of malformations including hydrops, eventration, wry neck, and brevicollis. PCB exposure was also found to produce histologic damage to liver and cutaneous tissue. Our data demonstrate that exposure of chicks to PCBs during development results in a retardation of growth, an increased incidence of malformations, and histopathologic damage.

Changes in urinary excretion of endothelin-1-like immunoreactivity in patients with testicular cancer receiving high-dose cisplatin therapy.

To assess the value of endothelin-1 (ET-1) in estimating renal injury in patients receiving high doses of cisplatin, urinary excretion of ET-1-like immunoreactivity (U-ET-1), beta 2-microglobulin (U-beta 2-MG), and N-acetyl-beta-d-glucosaminidase (NAG) were measured before, 1 week after, and 2 weeks after the administration of cisplatin in eight patients with testicular cancer (mean age, 33.3 years). Levels of U-ET-1/creatinine (Cr) during and 1 week after cisplatin treatment were significantly higher than before cisplatin treatment. There were no differences in U-ET-1/Cr levels during, 1 week after, and 2 weeks after cisplatin treatment. The level of U-beta 2-MG/Cr during cisplatin treatment was significantly higher than levels before, 1 week after, and 2 weeks after treatment. However, there were no differences in U-beta 2-MG/Cr levels before, 1 week after, and 2 weeks after cisplatin treatment. The level of U-NAG/Cr during cisplatin treatment was higher than levels before, 1 week after, and 2 weeks after treatment; U-NAG/Cr during cisplatin treatment was higher than levels before, 1 week after, and 2 weeks after treatment; U-NAG/Cr gradually decreased after cisplatin treatment. Among the three parameters, only U-ET-1/Cr maintained a higher level after cisplatin treatment. The U-beta 2-MG/Cr level returned most rapidly to normal after cisplatin treatment. Although U-ET-1/Cr did not show any significant correlation with U-NAG/Cr (r = 0.282, P = NS), it showed a significant correlation with U-beta 2-MG/Cr (r = 0.454, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary endothelin-1-like immunoreactivity in young male patients with testicular cancer treated by cis-platinum: comparison with other urinary parameters.

1. Urinary excretion of endothelin-1-like immunoreactivity and urinary excretion of other parameters (beta 2-microglobulin, N-acetyl-beta-D-glucosaminidase and microalbumin) were measured before, within 1 week after and 2 weeks after the administration of cis-platinum in five young male patients with testicular cancer (mean age 33.0 years) and were compared. 2. Urinary endothelin-1-like immunoreactivity/creatinine during, 1 week after, and 2 weeks after cis-platinum treatment was significantly higher than before cis-platinum. There was no difference in urinary endothelin-1-like immunoreactivity/creatinine during, 1 week after and 2 weeks after cis-platinum. 3. Among the four parameters, urinary endothelin-1-like immunoreactivity/creatinine showed the highest level after cis-platinum treatment. Urinary beta 2-microglobulin/creatinine most rapidly returned to normal levels after cis-platinum. 4. Although urinary endothelin-1-like immunoreactivity/creatinine did not show any significant correlations with urinary N-acetyl-beta-D-glucosaminidase (r = 0.291, not significant) or urinary microalbumin/creatinine (r = 0.076, not significant), it showed a significant correlation with urinary beta 2-microglobulin/creatinine (r = 0.475, P < 0.05). 5. These results suggest that endothelin-1 may be a sensitive urinary parameter in detecting cis-platinum-induced renal tubular injury.

Measurement of urinary endothelin-1-like immunoreactivity and comparison with other urinary parameters in patients with primary vesicoureteral reflux. A preliminary report.

By using a radioimmunoassay specific for endothelin-1 (ET-1), we measured urinary excretion of ET-1-like immunoreactivity in 63 spot urine samples of 48 patients with primary vesicoureteral reflux (VUR). Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), beta 2-microglobulin (beta 2-MG), microalbumin (Alb), and creatinine (Cr) were also measured. There was no significant correlation in any pairs of ET-1 and NAG, ET-1 and beta 2-MG or ET-1 and Alb. In patients with grade 2, grade 3, and grade 4 VUR, urinary ET-1/Cr was significantly higher than normal (p < 0.05). Comparing the grade of reflux according to the International Classification with urinary ET-1/Cr, the ratio of more than normal urinary ET-1/Cr increased in proportion to the grade of reflux, but it conversely decreased in grade 5. In conclusion, urinary ET-1 may be an indicator of distal renal tubular or collecting duct injury in patients with primary VUR.

Simultaneous measurement of cystometry and diuresis renography during full and empty bladder in neurogenic bladder patients: a preliminary report.

In 9 patients (4 male and 5 female; mean 24.7 years old) with neurogenic bladder dysfunction, diuresis renography and water filling cystometry were simultaneously monitored with a full and empty bladder, and findings of diuresis renography with a full bladder were compared with those of diuresis renography with an empty bladder. According to O'Reilly's classification, findings of diuresis renography with a full bladder were significantly worse than those of diuresis renography with an empty bladder, regardless of cystometry patterns and bladder compliances. Although every kidney without hydronephrosis on intravenous pyelogram showed normal diuresis renography with an empty bladder, some kidneys with hydronephrosis showed an obstructive pattern even with an empty bladder. These results suggest that in some patients with neurogenic bladder dysfunction, deterioration of the upper urinary tract occurs more easily with a full than with an empty bladder, regardless of bladder pressure and bladder compliance.

Changes in urinary excretion of endothelin-1-like immunoreactivity before and after unilateral nephrectomy in humans. Comparison with other urinary parameters and unilateral adrenalectomy.

To assess the value of endothelin-1 (ET-1) as a parameter of renal functional overload and the process of hyperfiltration, urinary excretion of ET-1-like immunoreactivity (U-ET-1) and urinary excretion of other parameters [beta 2-microglobulin (beta 2-MG), N-acetyl-beta-D-glucosaminidase (NAG), albumin (Alb) and 24-hour creatinine clearance (Ccr24)] were measured before, 1, 2 and 7 days after unilateral nephrectomy in 15 patients with unilateral renal lesions (renal cell cancer, renal pelvic cancer, ureteral cancer and renal tuberculosis) and after unilateral adrenalectomy in 5 patients with adrenal lesions. In the nephrectomy group (NX), Ccr24 significantly decreased at 1 and 2 days after the operation, but the other 4 parameters significantly increased after the operation as compared to preoperative values. In the adrenalectomy group (ADX), neither Ccr24 nor ET-1/Cr showed any change after the operation, but the other 3 parameters (beta 2-MG/Cr, NAG/Cr and Alb/Cr) significantly increased. Comparing the NX and ADX groups, both ET-1/Cr and beta 2-MG/Cr in NX were significantly higher than in ADX after the operation. On the other hand, neither NAG/Cr nor Alb/Cr in NX differed from values in ADX. Comparing ET-1/Cr and other parameters, only beta 2-MG showed a positive significant correlation. These results suggest that U-ET-1 may be an indicator of functional overload of the kidney.