Michael C. Gerald

Effect of Age on Cholinergic Muscarinic Responsiveness and Receptors in the Rat Urinary Bladder

The effects of age on urinary bladder responsiveness to muscarinic agonists and on the Bmax and Kd of the binding of [3H]quinuclidinyl benzilate (QNB) to muscarinic receptors of the bladder were studied. Bladder bodies and bases were isolated from Fischer 344 rats, ages seven, 16 and 27 months. No age-dependent change in maximum KCl-elicited isotonic contractions was observed in either bladder region. The bladder base showed an age-dependent increase in the maximum contractions (Emax) elicited by muscarinic agonists. The Emax values for bladder bases from rats 27 months of age were 44 per cent, 58 per cent and 76 per cent greater than those from rats seven months of age for acetylcholine, bethanechol and oxotremorine, respectively. No such alteration in responsiveness was observed in the bladder body with age. There were no age-related changes in ED50 values for the three agonists in either bladder region. Analysis of [3H]QNB binding in the bladder base demonstrated a modest 18 per cent increase in the Bmax (fmol./mg. tissue) from seven to 16 months and a significant 39 per cent decrease from 16 to 27 months. In the bladder body, the Bmax progressively increased by 25 per cent from seven to 27 months. The Kd values of [3H]QNB did not change with age in either region. The data demonstrate that the age-related increase in the responsiveness of the bladder is regionally specific and cannot be explained by a change in the number or affinity of muscarinic receptors.

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.

Age-related changes in sensitivity of rat urinary bladder to atonomic agents

Experiments were done to determine if age-related changes occur in autonomic regulation of rat urinary bladder. The maximum contractile responses to acetylcholine were 63% and 15% greater in isolated bladders from 29-month and 17-month animals, respectively, as compared to 7-month animals. The amounts of [3H]quinuclidinyl benzilate bound to membrane preparations were 46% and 7% greater. In contrast, no age-related changes were observed in phenylephrine-induced contraction or in isoproterenol-induced relaxation of bladder. Thus, the urinary bladder of aged rats appears to develop increased sensitivity to cholinergic stimuli because of an increase in the number of muscarinic cholinergic receptors.

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.

Streptozocin-induced Diabetes Affects Rat Urinary Bladder Response to Autonomic Agents

The response of the urinary bladder body and base to autonomic agents was studied instreptozocin (STZ)-diabetic rats. The bladder body region from 6-wk diabetic rats showed no changes in response to acetylcholine, phenylephrine, or isoproterenol. In contrast, the bladder base region showed a 39% increase in contractile response to acetylcholine and a 37% increased response to phenylephrine. In tissues from 47-wk diabetic animals, the bladder body showed a 51% increased contractile response to acetylcholine and a 37% increased relaxation response to isoproterenol. The bladder base showed a 66% increased contraction to acetylcholine. Thus, in the bladder base, enhanced responses to acetylcholine are detected soon after induction of diabetes and continue to increase as the diabetic state progresses. Moreover, in the same bladder region, an increase in responsiveness to alphaadrenergic stimuli occurs. In the bladder body, enhanced responses to cholinergic and to beta-adrenergic stimuli occur, but are only observed in a more chronic diabetic state. The data suggest that an effect associated with autonomic diabetic neuropathy of the urinary bladder is an increased postsynaptic responsiveness to cholinergic stimuli in both regions.

Effects of Diabetes and Diuresis on Contraction and Relaxation Mechanisms in Rat Urinary Bladder

Experiments were designed to gain information on the mechanisms leading to diabetic urinary bladder dysfunction. Bladders from control rats, animals subjected to 4–5 wk streptozocin-induced diabetes, and rats subjected to equivalent diuresis produced by 5% sucrose feeding were studied with an in vitro whole-bladder preparation and neurochemical measurements. The diuretic group was used to distinguish alterations produced by metabolic effects on nerve and muscle from those induced by prolonged periods of excessive diuresis. Diuresis alone explains many of the diabetes-induced effects, including decreased norepinephrine levels, postsynaptic supersensitivity for sympathetic regulation of bladder storage, decreased responsiveness to parasympathetic regulation of emptying, and enhanced prostaglandin F2α-induced contraction. Other diabetes-induced effects were not observed in the diuretic controls and are presumed to result from metabolic alterations associated with diabetes. These effects were decreases in norepinephrine uptake and in choline acetyltransferase activity, both markers of nerve terminal function. Thus, diuretic and metabolic factors appear to contribute to the early signs of parasympathetic and sympathetic neuropathy. In contrast, we found no evidence for loss of sensory nerve function in the diabetic bladder, at least at the organ level, because no diabetes- or diuresis-induced changes were observed in responsiveness to substance P or capsaicin.

Age-related change in α-adrenergic responsiveness of the urinary bladder of the rat is regionally specific

The effects of age on the responsiveness of the body of the urinary bladder and base of the bladder to alpha-adrenergic agonists were studied. Regions of the bladder were isolated from Fischer 344 rats, ages 7, 16, and 27 months. Maximum isotonic contractions elicited by potassium chloride (KCl) in both regions of the bladder were unaffected by age. In the bladder body there was an age-related increase in the maximum contraction elicited by phenylephrine, norepinephrine and clonidine. No such alteration in responsiveness was observed in the base of the bladder with age. The ED50 values of all three agonists were unchanged with age in both regions of the bladder. The pA2 values of prazosin and yohimbine were approximately 8.5 and 6.0, respectively, in the body of the bladder, and these values were not altered by age. Thus, it is concluded that an age-related increase occurs in the responsiveness of the body of the bladder to alpha-adrenergic activation and that these changes are mediated by alpha 1-adrenoceptors.

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 autonomie neuropathy. Four experimental conditions were compared: control, 4-wk-s t re ptozoc in-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 experiments, suggests that the galactose model does not produce irreversible functional neuropathies.

Comparative convulsant activity of various penicillins after intracerebral injection in mice

The convulsant effects of penicillin derivatives (penicillin G, Phenoxymethylpenicillin, penicillin O, ampicillin, methicillin) and cephaloridine have been evaluated after being given intracerebrally to mice. Penicillin G was most neurotoxic; ampicillin and Phenoxymethylpenicillin were one‐fifth as toxic. Incubation with penicillinase markedly reduced or eliminated the convulsant effects of penicillin G, Phenoxymethylpenicillin and ampicillin; this enzyme incubation did not alter the severity of seizures elicited by methicillin and cephaloridine. Diphenylhydantoin and phenobarbitone did not reduce penicillin G‐induced seizures. A non‐convulsant dose of leptazol potentiated the convulsions produced by the penicillins and cephaloridine. Injection volume, pH and the concentration of sodium and potassium did not produce behavioural changes within the ranges studied.

Studies on the effects of histaminergic agents on seizure susceptibility in mice.

The influence of pharmacological modifications of the functional activity of the central histaminergic system was studied on the susceptibility of mice to pentylenetetrazolinduced minimal (clonic) and maximal (tonic) seizures. Enhancement in the functional activity of the system by central administration of histamine or 4-methylhistamine or peripheral l-histidine loading failed to modify the risk of seizures. By contrast, reduction in histaminergic function was found to alter seizure susceptibility. Brocresine, an inhibitor of histamine synthesis, decreased and increased the risk of pentylenetetrazol-induced minimal and maximal seizures, respectively. Many, but not all, classical antihistamines (H1 antagonists) and metiamide (H2 antagonist) increased minimal seizure susceptibility after peripheral and intraventricular administration, respectively.