N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides: KATP potassium channel openers. Modifications on the western region.

A subset of antiandrogen compounds, the N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides 1, were found to activate ATP sensitive potassium channels (KATP) and represent a new class of potassium channel openers (PCOs). A structure-activity relationship was carried out on the western region of this series with the goal of obtaining an activator of the ATP sensitive potassium channel suitable for use in the treatment of urge urinary incontinence. In particular three large 4-(N-aryl) substituents, the (N-phenyl-N-methylamino)sulfonyl, benzoyl, and 4-pyridylsulfonyl moieties, yielded non-antiandrogen, KATP potassium channel openers (39, 41, and 64, respectively) that are bladder selective in an in vivo rat model that simultaneously measures bladder contractions, heart rate, and blood pressure. Substitutions of the aryl rings of 41 and 64 gave several derivatives that also display selectivity in the in vivo rat model; however, none appear to offer a substantial advantage over 41 and 64. The PCO activity of 41 and 64 resides in the (S)-(-) enantiomers. ZD6169, 41(S), has been selected into development for the treatment of urge urinary incontinence.

Effect of cromakalim on micturition function in rats.

Although many studies investigating the effect of cromakalim on bladder contractility exist, thus far, there are no published studies investigating its effect on micturition function in conscious rats. We measured the effect of cromakalim i.v. on urine output, frequency, volume of each micturition, and blood pressure in saline-diuresed and non-diuresed rats. In saline-diuresed rats cromakalim produced significant decreases in urine output (0.1 mg/kg, 32%; 0.3 mg/kg, 46%; 1.0 mg/kg, 68%) and average frequency (0.1 mg/kg, 36%; 0.3 mg/kg, 51%; 1.0 mg/kg, 70%) in the first 3 hours. At 3-6 hours after administration of cromakalim there were rebound increases in both urine output (0.1 mg/kg, 290%; 0.3 mg/kg, 373%; 1.0 mg/kg, 538%), and frequency (0.1 mg/kg, 147%; 0.3 mg/kg, 181%; 1.0 mg/kg, 314%) and by 6-12 hours the effects of cromakalim on micturition function were gone. Mean arterial pressure dropped to 50% of control immediately after cromakalim administration in saline-diuresed rats and began to return to control levels after 3 hours. Cromakalim produced similar results in non-diuresed rats. The decrease in urine output 0-3 hours after cromakalim administration may have been a consequence of cromakalim's profound decrease in blood pressure that occurred during that time.

Comparison of urinary bladder function in sexually mature and immature male and female rats.

Although studies exist using both male and female rats, there are virtually no studies that compare male and female bladder function. In this initial study, in-vivo and in-vitro urinary bladder function was investigated in two age groups of male and female rats (sexually immature and sexually mature). These studies compare in-vivo micturition behavior (water intake, urine output, frequency and volume per micturition); and in-vitro whole bladder function (bladder volume/pressure relationships, the ability of the in-vitro bladders to generate pressure and empty in response to bethanechol and field stimulation). The results can be summarized as follows: 1) The 24 hour water intake, urine output, and volume per micturition for the mature male rats was significantly greater than that of the mature females with no significant differences among the immature females, mature females, or immature males. 2) There were no significant differences in the frequency of micturition between the 4 groups. 3) Although the average plateau pressures (cystometrograms) of the immature and mature female bladders were greater than that of the immature and mature male bladders, the compliance was similar for all groups. 4) The maximum pressure response of the mature female bladder was significantly greater than pressures generated by bladders in the other three groups; there were no age or sex related differences in the bethanechol log ED50 values. 5) There were no age or sex-related differences in the bethanechol log ED50 values or maximal expulsion responses. 6) Field stimulated bladders from mature animals generated significantly greater intravesical pressures than bladders from immature animals, but, there were no significant differences in maximal pressures attained between mature male and female bladders nor between immature male and female bladders. In conclusion, micturition behavior, and the maximal pressure response to bethanechol changed dramatically with sexual maturity. These results are consistent with the idea that estrogen and other hormones may have a marked influence on bladder function and micturition behavior.

Functional and pharmacological effects of ureteral diversion.

Urinary diversion is the final therapeutic approach in several bladder pathologies including selected cases of bladder cancer, neurogenic bladder, and painful bladder syndrome. While there have been a few experimental studies on the effects of urinary diversion and subsequent undiversion on bladder capacity, the pharmacological changes occurring with diversion and undiversion have not yet been investigated. Our primary objectives were to determine the functional and pharmacological alterations in the defunctionalized bladder and the effects of refunctionalization on these changes. Thirty-two male adult canines weighing 15 to 20 lb. were used for this study. Three groups of dogs were urine diverted for one, three, and six month durations (four dogs per group). Another three groups of dogs were urine diverted for one, three, and six months (four dogs per group); these dogs were then undiverted for three months. Six control animals received either one or two sham operations at one, three, and six months. In all groups the functional and contractile characteristics of the bladders were assessed by urodynamics and in vitro contractile studies; bladder weight and muscarinic receptor density were also measured. Intravesical capacities determined by in vivo cystometry were reduced significantly to 74%, 63%, and 47% of control values at one, three, and six months respectively after urinary diversion. Bladder capacity returned to above normal levels in bladders diverted and then subsequently undiverted. Similarly, the compliance and bladder weight of the diverted bladders were significantly less than control, while diverted bladders subsequently undiverted were similar to controls. Maximal bladder contractility in response to bethanechol stimulation was less in diverted bladders compared to control. This decrease in contractility was accompanied by a decrease in muscarinic receptor density. After undiversion maximal bladder contractility to bethanechol reached control levels; this was accompanied by a parallel increase in muscarinic receptor density to control values. There was no effect of diversion or undiversion on the maximal response of bladder strips to methoxamine stimulation. Thus, a bladder that has been diverted (defunctionalized) for a period of time showed decreases in capacity, compliance, and contractility to muscarinic stimulation along with a decrease in muscarinic receptor density. All of these parameters were restored after refunctionalization of the bladder.

Effects of age on urinary bladder function in the male rat.

In a previous study we investigated the effects of age on the micturition characteristics and bladder function of male Fischer rats ages five to seven, 16 to 18 and 22 to 24 months. The 24 hr. water intake and urine output increases significantly with age; 22 to 24 month rats showed a 39% increase in water intake and a 93% increase in urine output compared to five to seven month rats. The intravesical pressure at micturition is 100% greater in 22 to 24 month and 16 to 18 month rats compared to five to seven month old rats with no age-related change in bladder volume at micturition. In the present study, in vitro bladder capacity did not differ between the three age groups although the average plateau pressure significantly decreased with advancing age. Using the isolated whole bladder model, the contractile response to the autonomic agonists bethanechol, phenylephrine, and isoproterenol did not change significantly with age. Similarly, there were no age-related changes in the response of the bladder to non-autonomic drugs (histamine, oxytocin, serotonin, substance P, and PGF2 alpha) except for PGF2 alpha which produced an age-related increase in the maximum bladder contraction. In summary, while in vivo micturition clearly changes with age, the in vitro contractility of the bladders to autonomic agents did not. Therefore, age related differences in micturition would be related primarily to the changes in neuronal innervation and central control of micturition rather than alterations in the contractility of the bladder. In addition, these studies show the importance of correlating in vivo bladder function (micturition frequency and volume, cystometry and urodynamics) with in vitro contractile and functional studies.

Pharmacological comparison of the isolated whole urethra model to urethral strip methodology.

Urethral strips provide a sensitive method for quantitative pharmacology; however, it is not clear if the physiological response of the whole urethra to pharmacological agents can be extrapolated from these data. The intent of this study was to investigate whether the linear contraction of strips is consistent with the ability of the intact urethra to alter resistance to flow. To measure the effect of drugs on the ability of the intact urethra to alter resistance to flow in the absence of endogenous influences, we developed an in vitro whole rabbit urethra model. We characterized the effect of norepinephrine in the absence and presence of alpha-adrenergic antagonists (prazosin, yohimbine, and LY253352) on intraurethral pressure which was measured during constant saline infusion using a Statham pressure transducer. We compared this to the contractile function of urethral strips. The pA2 values for alpha-adrenergic antagonists, LY253352, prazosin, and yohimbine were 8.17 +/- 1.5, 6.49 +/- 1.2, and 5.58 +/- 0.9, respectively, as determined in the whole urethra. The pA2 values for these antagonists determined in urethral strips were as follows: LY253352 8.72 +/- 1.2; prazosin 7.52 +/- 1.5, and yohimbine 5.6 +/- 0.84. Pharmacologically, the whole urethra model and isolated strips appeared to respond similarly to alpha-adrenergic agonists and antagonists, and thus both models would be suitable for pharmacological studies on the normal urethra.

Effect of contractile activity on muscarinic receptor density and the response to muscarinic agonists.

Autonomic receptor density can be modulated by alterations in neuronal activity over a relatively short period of time (hours). The current study investigates whether increased in vivo stimulation of urinary bladder smooth muscle can alter muscarinic receptor density and response to muscarinic stimulation. A high degree of reflex stimulation of the urinary bladder (rabbits) was initiated by stricture of the external urethra. Intravesical pressure and intra-abdominal pressure were monitored continuously over a 4-hr time period. At the end of the 4-hr period, the rabbits were sacrificed and isolated strips of bladder body were either mounted in isolated smooth muscle baths for contractile studies or frozen and stored in liquid nitrogen for muscarinic receptor analysis. These studies demonstrated that over 4 hr of urethral stricture there was a significant reduction in muscarinic receptor density from a Bmax of 34 +/- 3.4 fmol/mg of protein in control bladder strips to 22 +/- 2.4 fmol/mg of protein in the experimental group. In association with the decreased muscarinic receptor density, there was a significant and selective decrease in the contractile response to muscarinic stimulation. Similar to the in vivo studies, repetitive field stimulation of in vitro strips resulted in a significant decrease in muscarinic receptor density and a significant and selective decrease in the contractile response to muscarinic stimulation. The results from these studies indicate that muscarinic receptor density, and response to muscarinic stimulation, can be modulated over a relatively short period of time by alterations in the level of neuronal stimulation.

Diabetes and diuretic-induced alterations in function of rat urinary bladder.

Studies were done to characterize the bladder dysfunction associated with diabetes mellitus and to distinguish between changes occurring from increased diuresis and autonomic neuropathy. Four experimental conditions were compared: control, 4-wk-streptozocin-induced diabetes, sucrose feeding (diuretic), and galactose feeding (diuretic and sugar alcohol). A 10-fold increase in urine output and 25-50% increases in bladder weight, protein content, and DNA content were observed in all noncontrol treatment groups. Compliance properties were studied by measuring the intravesicular pressure as the bladder was infused with buffer in vitro. All treated bladders exhibited a reduction in plateau pressure and an increase in fluid capacity. Thus, diuresis results in an increased bladder size, which correlates with an alteration of compliance properties. Nervous system control in anesthetized rats was examined by monitoring contractions as the bladder was infused with buffer. Three distinct patterns of response were observed: normal, diabetic, and diuretic (galactose and sucrose treatments). The difference between responses in diuretic and diabetic animals suggests the presence of a diabetes-induced alteration in nerve regulation of the bladder. Reserpine pretreatment of control or diuretic models produced marked changes in the pattern of contractions, whereas pretreatment of diabetic rats had only modest effects. This suggests that diabetic bladders were lacking sympathetic control before the drug treatment. When rats treated for 4 wk with galactose were removed from this diet for 4 wk before testing, the bladders responded similarly to controls. This observation, coupled with the fact that galactose did not produce the same response as diabetes in the in vivo experiment, suggests that the galactose model does not produce irreversible functional neuropathies.

Effect of age on in vivo urinary bladder function in the rat.

The effects of age on micturition in male Fischer 344 rats, ages five to seven, 16 to 18 and 22 to 24 months, were studied. The 24 hr. water intake, 24 hr. urine output, frequency and volume of each micturition were obtained from rats housed individually in metabolic cages. Intravesical pressure and volume at which the micturition contraction occurred were evaluated using natural-fill cystometry. The 24 hr. water intake and urine output increased significantly with advancing age; 22 to 24 months rats showed a 39% increase in water intake and a 93% increase in urine output compared to five to seven month rats. The increase in urine output observed in the 22 to 24 month old rats was manifested by a 95% increase in volume per micturition and a 52% increase in frequency of micturition compared to five to seven month old rats. The pressure at micturition (PAM) was 100% greater in 22 to 24 and 16 to 18 month old rats compared to five to seven month old rats with no age-related difference in bladder volume at micturition (BVM). These studies demonstrate that in vivo micturition changes with age in the male F344 rat. Although there were no overt urological dysfunctions observed in the aging rats, the alterations in function would indicate that there were changes in either the mechanisms controlling micturition, or changes in the musculature itself. These possibilities will be the subject of further investigations.