Masaki Yoshida

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.

Age-dependent Alterations in beta-adrenergic Responsiveness of Rat Detrusor Smooth Muscle

The relaxant effects of norepinephrine (NE, 10(-7) to 10(-4) M.) and isoproterenol (ISO, 10(-9) to 10(-4) M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10(-6) to 3 x 10(-5) M.), dibutyryl cyclic AMP (DBcAMP, 10(-4) to 3 x 10(-3) M.) and cholera toxin (10 micrograms/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 micrograms) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [125I]iodopindolol ([125I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.

The reversal effect of insulin on diabetes-induced alterations in beta adrenergic and muscarinic receptors in rat prostate.

Previous studies from our laboratory demonstrated that 8 weeks after the induction of diabetes by the administration of streptozotocin (STZ) there was a downregulation of beta adrenergic and muscarinic cholinergic receptors in rat prostate, and that early insulin treatment (started 3 days after the onset of diabetes) prevented these alterations from occurring. In the present study, the effects of later insulin treatment (started 8 weeks after the onset of diabetes) on the reversibility of diabetes-induced alterations in beta adrenergic and muscarinic receptors in rat prostate were investigated. Three groups of rats were maintained for 16 weeks: 1) diabetics, 2) insulin-treated diabetics (subcutaneously injected with 5 to 8 U per day starting 8 weeks after the onset of diabetes) and 3) age matched controls. Binding studies with [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) showed a significantly lower density of beta adrenergic and muscarinic cholinergic receptors in the diabetic rat prostate than in prostate from either controls or insulin-treated diabetic animals. Inhibition of [3H]DHA binding by isoproterenol, a beta adrenergic agonist, and binding of [3H]QNB by carbachol, a muscarinic agonist, indicated the presence of low and high affinity agonist binding sites for each receptor. The relative proportion of high affinity to total binding sites as well as the low and high affinity constants were similar in all groups. These data indicate that insulin treatment, begun 8 weeks after the onset of diabetes, can reverse the diabetes-induced downregulation of both beta adrenergic and muscarinic cholinergic receptors in STZ-diabetic rat prostates.

Reversibility of Diabetes- and Diuresis-Induced Alterations in Rat Bladder Dome Muscarinic Receptors

Previous studies from our laboratory demonstrated that 8 weeks of streptozocin (STZ)-induced diabetes and sucrose-fed diuresis resulted in increases in the density of muscarinic receptors in rat bladder dome and that early insulin treatment (started 3 days after the onset of diabetes) prevented the diabetes-induced upregulation (J Pharmacol Exp Ther 248:81–88, 1989; Diabetes 40: 1150–1156, 1991; J Urol 147:760–763, 1992). To determine whether diabetes- and diuresis-induced alterations in muscarinic receptors in rat bladder dome are reversible, we administered insulin (beginning 8 weeks after the onset of diabetes) or removed sucrose from drinking water of diuretic rats (beginning 8 weeks after the onset of diuresis). Five groups of rats were maintained for 16 weeks: 1) STZ-induced diabetic rats (65 mg/kg intravenously); 2) insulin-treated diabetic rats (5–8 U/day insulin subcutaneously beginning 8 weeks after the onset of diabetes); 3) sucrose-fed diuretic rats (5% sucrose in drinking water throughout 16 weeks); 4) sucrose-removed rats (sucrose withdrawn from drinking water after 8 weeks of the sucrose-induced diuretic state); and 5) age-matched control rats. Radioligand receptor binding experiments with [3H]quinuclidinyl benzilate showed an increase in the density of muscarinic receptors in bladder dome of diabetic and sucrose-fed rats compared with age-matched control rats. Removing the 5% sucrose from the drinking water of diuretic rats reversed the increased water intake and urine output, decreased the bladder hypertrophy that accompanied the diuretic state, and corrected the upregulation of the muscarinic receptors. Late insulin treatment also improved (to a lesser extent than removing the sucrose) the bladder enlargement, polydipsia, polyuria, and the muscarinic receptor upregulation in diabetic rats. Pharmacological evaluation indicated that the induction of diabetes did not affect muscarinic subtype specificity but did result in an alteration in muscarinic receptor-G protein coupling in rat bladder dome.

Muscarinic receptors in diabetic rat prostate

To investigate the effects of experimentally-induced diabetes on prostatic muscarinic cholinergic receptors, the binding characteristics of [3H]quinuclidinyl benzilate ([3H]QNB) to prostatic membrane particulates were examined in four groups of rats: control, diabetic, diabetic insulin treated, and diabetic myo-inositol treated. Diabetes was induced by i.v. injection of streptozotocin (STZ), 65 mg/kg. Diabetic and diabetic myo-inositol-treated rats had hyperglycemia, hypoinsulinemia, glucosuria, polydipsia, and polyuria as well as significantly smaller prostates and lower body weights compared to control and diabetic insulin-treated animals. The densities of muscarinic receptors (Bmax) as determined by saturation studies with [3H]QNB in the prostatic plasma membranes of control, diabetic, diabetic insulin-treated and diabetic myo-inositol-treated rats were 80 +/- 8, 51 +/- 5, 78 +/- 3, and 47 +/- 7 fmol/mg of protein, respectively. [3H]QNB binding to muscarinic receptors was inhibited by muscarinic antagonists with the following rank order of Ki values: atropine much less than pirenzepine less than AF-DX 116. The pharmacological profile of the muscarinic receptors was similar in all groups examined and was consistent with the predominance of the M3 muscarinic receptor subtype in prostatic membrane particulates. Our data indicate that STZ-induced diabetes caused a variety of abnormalities including a down-regulation in the density of M3 muscarinic receptors in the rat prostate and that insulin, but not myo-inositol could prevent the development of these abnormalities.

Evidence for the presence of regional differences in the calcium antagonist receptors in lower urinary tract smooth muscle

Summary(+)-[3HPN 200-110 (a dihydropyridine calcium channel antagonist) was utilized to characterize calcium channel binding sites in rabbit bladder dome, bladder base, and urethra. Specific binding of (+)-[3H]PN 200-110 to membrane particulates was saturable, reversible, linear to protein concentration, and of high affinity. The density of (+)-[3H]PN 200-110 binding sites (Bmax values in fmol/mg of protein) and the affinity constants for (+)-[3H]PN 200-110 (KD value in pM) in urethra, bladder dome and bladder base were 64.1 ± 7.8 and 179±31; 21.9±3.0 and 213±36; and 18.8±4.2 and 140±28, respectively. Agonists and antagonists inhibited (+)-[3H]PN 200-110 binding with Ki values in the following rank order: nitrendipine < nifedipine < niguldipine ≪ Bay K 8644 ≪ verapamil. Although carbachol-induced contractile responses were 20–30 times smaller in muscle strips from urethra than from bladder base or bladder dome, KCl-induced contractions were only 3–4 times smaller in urethra than in bladder tissues. Nifedipine inhibited carbachol-induced contractions in urethra, bladder dome, and bladder base by 76%, 64%, and 60%, respectively, and completely inhibited KCl-induced contractions in all three tissues. IC50 values for nifedipine inhibition of both carbachol- and KCl-induced contractions were significantly smaller in urethra than in bladder base or bladder dome. Nitrendipine, niguldipine and verapamil inhibited urethral contractions induced by carbachol and KCl to the same degree as did nifedipine. The IC50 values, obtaines from functional studies, for calcium channel antagonists were in good agreement with Ki values obtained from binding studies. BAY K 8644, a calcium channel agonist, increased both KCl- and carbachol-induced contractions and potentiated CaCl2-induced contractions in K+-depolarizing media in urethra but not in bladder dome or bladder base. Our data indicate that the density of (+)-[3H]PN 200-110 binding sites is higher in the urethra than in the bladder dome or bladder base and that the urethra is functionally more sensitive to dihydropyridine agonists and antagonists than is either the bladder dome or bladder base.

Effect of insulin and dietary myoinositol on muscarinic receptor alterations in diabetic rat bladder

Previous studies from our laboratory demonstrated that there is an up-regulation of muscarinic receptor density in the bladder dome of the 8-wks diabetic rat compared to control. To determine whether the changes observed in receptor density can be corrected by insulin or dietary myoinositol, five groups of rats were maintained for eight weeks: control (C), diabetic (D), diabetic insulin-treated (DI), diabetic myoinositol-treated (DMI), and control myoinositol-treated (CMI). Diabetes was induced by i.v. injection of 65 mg./kg. of streptozotocin. D and DMI animals were smaller, had higher serum glucose and lower serum insulin levels, higher water intakes and urine outputs, and larger bladder domes than the other groups. The density of the muscarinic receptors measured by radioligand receptor binding assays using [3H]quinuclidinyl benzilate were (in fmol./mg. protein): C, 88 +/- 13; D, 176 +/- 19; DI, 94 +/- 5; DMI, 158 +/- 8; CMI, 112 +/- 10. These data indicate that insulin, but not myoinositol treatment normalized diabetes induced abnormalities observed in the general features of streptozotocin-injected rats and prevented the diabetic-induced upregulation of bladder dome muscarinic receptors.

Diabetes-induced alterations in the properties of muscarinic cholinergic receptors in rat vas deferens

Muscarinic cholinergic receptors were identified and characterized by radioligand receptor binding assay using [3H]quinuclidinyl benzilate (QNB) in rat vas deferens membrane particulates of three experimental groups: 1) 8-week diabetic, 2) 8-week diabetic insulin-treated and 3) age-matched control. Diabetes was induced by the intravenous injection of 65 mg./kg. streptozotocin (STZ). The density of muscarinic receptors (Bmax values), as determined by saturation experiments with [3H]QNB, was demonstrated to be higher in the vas deferens of diabetic rats than in the vas deferens of control and diabetic insulin-treated rats. The equilibrium dissociation constants (KD values), however, were similar in all three groups. Muscarinic cholinergic antagonists competed with [3H]QNB binding sites in the vas deferens membrane particulates with the following rank order of Ki values: atropine < methoctramine < or = 4-DAMP < AF-DX 116 < HHSiD < pirenzepine = pfHHSiD. The pharmacological profile of muscarinic receptors was similar in all three groups. Additional pharmacological studies showed a similar rank order of Ki values for vas deferens, bladder dome and heart, but this rank order was significantly different in cerebral cortex and prostate. This is consistent with the predominance of the M2 muscarinic cholinergic receptor subtype in the rat vas deferens. It is concluded that STZ-induced diabetes causes an upregulation of muscarinic cholinergic receptor density in the rat vas deferens that can be prevented by the administration of insulin.

Experimental Diabetes Upregulates the Expression of Uretereral Endothelin Receptors

We investigated the binding characteristics of endothelin (ET) receptors in the ureters of rats with experimentally induced diabetes and diuresis. Receptor binding experiments demonstrated an upregulation in the expression of [125I]ET-1 binding sites in the diabetic rat ureter but not in the diuretic rat ureter. ET-1, ET-3, IRL 1620, and BQ 610 inhibited [125I]ET-binding to the rat ureter consistent with the predominance of ETA receptors in these tissues. The subtype specificity of ET receptors in ureteral tissues was confirmed with inhibition data obtained from cloned human ETA and ETB receptors.

Identification of potential therapeutic targets in hypertension-associated bladder dysfunction

To investigate differential gene expression profiles in the bladder of spontaneously hypertensive rat (SHR), as the underlying mechanisms involved in hypertension‐associated bladder dysfunction remain to be clarified.