Nitric oxide production within rat urothelial cells.

PURPOSE: Recent studies have suggested that nitric oxide (NO) synthase (NOS) may be localized in the urothelium of the proximal part of the mammalian ureter. We investigated endogenous NO production in the proximal half of the rat ureter, localized its cellular source, characterized the NOS isoforms involved and assessed the impact of NO on ureteral motility. MATERIALS AND METHODS: Direct detection of NO production was performed on primary cultures of living rat ureteral cells with the fluorescent indicator diaminofluorescein. Cultures were incubated with the NO precursor L-arginine or the NOS inhibitors L-NAME (N-nitro-L-arginine-methyl ester) and 1400W. NOS expression was determined by immunofluorescence and Western blot analysis. The functional effects of NO donors were assessed on isolated ureters. RESULTS: Significant basal NO production was demonstrated by the high fluorescence level detected in diaminofluorescein treated cell cultures. NO production was strictly limited to urothelial cells since no fluorescence was seen in smooth muscle cells. Pretreatment with L-NAME or 1400W resulted in a significant decrease in fluorescence. Constitutive and inducible NOS isoforms were detected in urothelial cultured cells and in lysates of the urothelial layer. NO donors inhibited in a concentration dependent manner the agonist induced contractile activity of isolated ureters. CONCLUSIONS: These results suggest that NO production stems from the urothelium and the NO pathway inhibits contractile activity in the proximal half of the rat ureter. Hence, the nitrergic pathway may be an important target for drugs producing relaxation of the mammalian ureter.

Acid prohormone sequence determines size, shape, and docking of secretory vesicles in atrial myocytes.

How vesicles are born in the trans-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 microm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (r=0.71, P<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu(23,24)-->gln(23,24)) change size and shape-but not velocity-of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate-docking or not docking-at the plasma membrane. The full text of this article is available at http://www.circresaha.org.

Pituitary adenylate cyclase-activating polypeptide activates K(ATP) current in rat atrial myocytes.

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K(+) (K(ATP)) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC(50) = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC(1) (> or =2 isoforms), VPAC(1), and VPAC(2). PACAP-38 dose dependently activates the whole cell atrial K(ATP) current with EC(50) = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 +/- 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H(89) (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K(ATP) current to 17.8 +/- 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K(ATP) current. A protein kinase C (PKC) inhibitor peptide (50 micromol/l) in the pipette reduces the PACAP-38-induced K(ATP) current to 33 +/- 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC(1), VPAC(1), and VPAC(2) are present in atrial myocytes; and 2) PACAP-38 activates the atrial K(ATP) channels through both PKA and PKC pathways.

Sulfonylurea receptor ligands modulate stretch-induced ANF secretion in rat atrial myocyte culture.

Stretch-induced atrial natriuretic factor (ANF) secretion was studied in cultures of neonate atrial appendage myocytes. Stretch, applied for 40 min by hypotonic swelling, increased the mean area of 44 individually imaged myocytes by 4.8-8.8% (P < 0.0001) at 6 min and by 2.3-6.2% (P < 0.05) at 35 min. Stretch increased immunoreactive ANF release by 42% (P < 0.05) from a baseline of 315 pg/ml. The ATP-sensitive K(+) (K(ATP))-channel blocker tolbutamide (100 micromol/l) increased the stretch-stimulated release to 84% (P < 0.01) over baseline, whereas lower concentrations (1, 10, and 30 micromol/l) had no stimulatory effect. The K(ATP)-channel opener diazoxide (0.1, 1, 10, 30, and 100 micromol/l) inhibited stretch- plus tolbutamide-stimulated ANF release in a concentration-dependent manner, with IC(50) = 2.2 micromol/l, although 100 micromol/l diazoxide did not reduce the increase in mean cell area. The stretch-stimulated K(ATP) current, monitored in 82 whole cell recordings with sulfonylurea receptor (SUR) ligands, was inversely correlated with the stretch-induced ANF release (r(2) = 0.79, P < 0. 0001). In the absence of stretch, the K(ATP) current had no relationship with baseline ANF release, and baseline ANF release was not affected by the K(ATP)-channel modulators. The results show that SUR ligands that open K(ATP) channels inhibit stretch-induced ANF release in atrial myocytes, in correlation with the stretch-activated K(ATP) current. The subcellular site of action of the SUR ligands-plasmalemma or intracellular organelles-remains to be determined.

Role of endothelin receptor subtypes in volume-stimulated ANF secretion.

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.

A novel K(ATP) current in cultured neonatal rat atrial appendage cardiomyocytes.

The functional and pharmacological properties of ATP-sensitive K(+) (K(ATP)) channels were studied in primary cultured neonatal rat atrial appendage cardiomyocytes. Activation of a whole-cell inward rectifying K(+) current depended on the pipette ATP concentration and correlated with a membrane hyperpolarization close to the K(+) equilibrium potential. The K(ATP) current could be activated either spontaneously or by a hypotonic stretch of the membrane induced by lowering the osmolality of the bathing solution from 290 to 260 mOsm/kg H(2)O or by the K(+) channel openers diazoxide and cromakalim with EC(50) approximately 1 and 10 nmol/L, respectively. The activated atrial K(ATP) current was highly sensitive to glyburide, with an IC(50) of 1.22+/-0.15 nmol/L. Recorded in inside-out patches, the neonatal atrial K(ATP) channel displayed a conductance of 58.0+/-2.2 pS and opened in bursts of 133.8+/-20.4 ms duration, with an open time duration of 1.40+/-0.10 ms and a close time duration of 0.66+/-0.04 ms for negative potentials. The channel had a half-maximal open probability at 0.1 mmol/L ATP, was activated by 100 micromol/L diazoxide, and was inhibited by glyburide, with an IC(50) in the nanomolar range. Thus, pending further tests at low concentrations of K(ATP) channel openers, the single-channel data confirm the results obtained with whole-cell recordings. The neonatal atrial appendage K(ATP) channel thus shows a unique functional and pharmacological profile resembling the pancreatic beta-cell channel for its high affinity for glyburide and diazoxide and for its conductance, but also resembling the ventricular channel subtype for its high affinity for cromakalim, its burst duration, and its sensitivity to ATP. Reverse transcriptase-polymerase chain reaction experiments showed the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B subunits, a finding supporting the hypothesis that the neonatal atrial K(ATP) channel corresponds to a novel heteromultimeric association of K(ATP) channel subunits.

Effect of urea and detergents on the ability of human spermatozoa to penetrate zona-free hamster oocytes.

Washing of human spermatozoa with either BWW alone or with the same buffer containing 0.1 M urea, 0.005% SDS or 0.001% NP40 affected the penetration ability of the gametes into zona-free hamster oocytes to various degrees. In contrast, human spermatozoa washed with BWW buffer containing 0.3 M urea had an increased ability to fuse with the heterologous oocytes when compared to controls capacitated with BWW/BSA. Moreover, the presence or absence of BSA in the insemination medium did not further modify this enhanced penetration pattern. The BWW, BWW/0.1 M urea and BWW/SDS treatments apparently mimicked some of the in vitro capacitation properties of albumin-containing media; the BWW/0.3 M urea treatment overpowered the capacitation and acrosomal reaction extent obtained with BWW+BSA. In all samples the motility of the spermatozoa washed with BWW buffer alone or containing various additives (but no albumin) was significantly decreased if compared to the motility of semen samples washed with albumin containing media. However, each sperm sample behaved differently when exposed to a given buffer.

Oocyte penetration and acrosome reactions of human sperms. II: Correlation with other seminal parameters.

Washed sperm suspensions of fertile men and men consulting for infertility were evaluated for their ability to penetrate zona-free hamster eggs and for their ability to exhibit an acrosome reaction in vitro. Furthermore, the swelling of the spermatozoa under hypoosmotic conditions as indication of their membrane integrity was determined. In the group of fertile men and in the group of patients with normal spermiogram, significantly more acrosome reactions were observed than in the group of infertile men with abnormal spermiogram parameters (p less than 0.05). This difference was still more significant when men with a positive hamster penetration test (H.O.P. test) and men with a negative H.O.P. test were compared (p less than 0.005). However, within the groups the level of acrosome reactions after incubation appeared to be highly variable. In a second series of experiments, working with semen obtained during our in vitro fertilization program, we found that the fertilization of human ova does not seem to be dependent on a strong progress of the acrosome reaction. Finally, swelling of the spermatozoa in a hypoosmotic medium was weakly correlated (r = 0.70, p less than 0.01; n = 73) with trypan blue exclusion. No significant correlations with other semen parameters, hamster ova test included, were found.

Oocyte penetration and acrosome reactions of human spermatozoa. I: Influence of incubation time and medium composition.

Washed sperm suspensions were evaluated for their ability to penetrate zona-free hamster oocytes and to exhibit an acrosome reaction in vitro. The percentage of acrosome reactions (AR%) was found to increase with incubation time and increased bovine serum albumin (BSA) concentration. We also found, however, that a remarkably high number of live, unreacted spermatozoa persisted during prolonged incubation in sperm samples obtained from some fertile men. After 22 hrs incubation, the motility of the spermatozoa was higher when the medium was supplemented with 10% human cord serum instead of 3.0% BSA. Penetration rates in hamster oocytes were not significantly different with 10% serum or 3.0% BSA as medium-supplementation. The addition of human instead of bovine serum albumin did not apparently affect the oocyte penetration rate of the spermatozoa.