Kristian Waldeck

INTRAVESICAL OXYBUTYNIN FOR NEUROGENIC BLADDER DYSFUNCTION: LESS SYSTEMIC SIDE EFFECTS DUE TO REDUCED FIRST PASS METABOLISM

PURPOSE To unravel why intravesical oxybutynin is more effective and causes significantly fewer systemic side effects than oral oxybutynin in the treatment of neurogenic bladder dysfunction, we tested the hypothesis that the absorption and metabolism of oxybutynin are changed after intravesical instillation. MATERIALS AND METHODS A high-performance liquid chromatography assay was developed for both oxybutynin and its active metabolite, N-desethyl-oxybutynin. Plasma concentrations were quantified after intravesical (n = 11) and oral (n = 5) administration of oxybutynin in children under steady-state conditions. Pharmacokinetic parameters were calculated. RESULTS Oral administration of oxybutynin (0.2 mg./kg./dose) resulted in peak plasma concentrations for N-desethyl-oxybutynin which were 7.4 +/- 1.3 times higher than corresponding values for oxybutynin (n = 5). Also the AUC (area under the plasma concentration time curve) values were higher for N-desethyl-oxybutynin compared with those of oxybutynin, the ratio being 10.8 +/- 1.0 (n = 5). Intravesical instillation (0.2 mg./kg./dose), on the other hand, resulted in reduced metabolite generation and peak plasma concentrations for N-desethyl-oxybutynin which were in the same range as those for oxybutynin, the ratio being 1.2 +/- 0.1 (n = 11). The ratio for the AUC values for N-desethyl-oxybutynin and oxybutynin was 2.1 +/- 0.2 (n = 11). CONCLUSIONS The significantly lower AUC ratio of the N-desethyl metabolite over the mother compound, due to a reduced first pass metabolism, may explain the clinically relevant reduction of side effects that characterizes intravesical compared with oral oxybutynin therapy.

Studies on the effects of anandamide in rat hepatic artery

1 The effects of anandamide on K+ currents and membrane potential have been examined in freshly‐isolated smooth muscle cells from rat hepatic artery and the results compared with the effects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery. 2 In the presence of 0.3 mm l‐NOARG and 10 μm indomethacin, anandamide (0.1–100 μm) and endothelium‐derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01–10 μm) each relaxed endothelium‐intact segments of hepatic artery precontracted with phenylephrine. These effects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 μm). 3 The relaxant effects of anandamide were unaffected by a toxin combination (apamin plus charybdotoxin, each 0.3 μm) which abolishes EDHF relaxations and were essentially unchanged in endothelium‐denuded arteries. The relaxant effects of anandamide in endothelium‐intact arteries were significantly reduced in a physiological salt solution containing 30 mm KCl and abolished when the K+ concentration was raised to 60 mm. 4 Anandamide (10 μm), acetylcholine (1 μm, via release of EDHF) and levcromakalim (10 μm) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium‐intact arteries. However, when the endothelium was removed, the hyperpolarizing effects of both anandamide (10 μm) and acetylcholine were essentially abolished whereas those of levcromakalim (10 μm) were unaffected. 5 Under voltage‐clamp conditions, anandamide (10 μm) abolished spontaneous transient outward currents (STOCs) in freshly‐isolated single hepatic artery cells held at 0 mV but had no effect on the holding current at this potential. In current‐clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no significant change in basal membrane potential. 6 Anandamide (10 μm) abolished the iberiotoxin‐sensitive K+ current (IBK(Ca)) produced by caffeine and the corresponding hyperpolarizations generated by this xanthine derivative in current‐clamp mode. In contrast, anandamide had no effect on IBK(Ca) generated on exposure to NS1619 (30 μm). 7 It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide‐induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrine‐contracted vessels.

Mediators and mechanisms of relaxation in rabbit urethral smooth muscle.

Electrophysiological and mechanical experiments were performed to investigate whether the nitric oxide (NO)‐mediated relaxation of rabbit urethral smooth muscle is associated with a hyperpolarization of the membrane potential. In addition, a possible role for vasoactive intestinal peptide (VIP) and carbon monoxide (CO) as relaxant agents in rabbit urethra was investigated. Immunohistochemical experiments were performed to characterize the NO‐synthase (NOS) and VIP innervation. Possible target cells for NO were studied by using antisera against cyclic GMP. The cyclic GMP‐immunoreactivity was investigated on tissues pretreated with 1 mM IBMX, 0.1 mM zaprinast and 1 mM sodium nitroprusside. Intracellular recordings of the membrane potential in the circular smooth muscle layer revealed two types of spontaneous depolarizations, slow waves with a duration of 3–4 s and an amplitude of 30–40 mV, and faster (0.5–1 s), more irregular depolarizations with an amplitude of 5–15 mV. The resting membrane potential was 39±1 mV (n=12). Application of NO (30 μM), CO (30 μM) or VIP (1 μM) did not change the resting membrane potential. Both NO (1–100 μM) and VIP (1 nM–1 μM) produced concentration‐dependent relaxations amounting to 87±4% and 97±2% (n=6), respectively. The relaxant effect of CO (1–30 μM) amounted to 27±4% (n=5) at the highest concentration used. Immunohistochemical experiments revealed a rich supply of NOS‐immunoreactive nerve fibres in the smooth muscle layers. Numerous spinous cyclic GMP‐immunoreactive cells were found interspersed between the smooth muscle bundles, mainly localized in the outer layer. These cells had long processes forming a network surrounding the smooth muscle bundles. VIP‐immunoreactivity was sparse in comparison to NOS‐immunoreactive nerves. The rich supply of NOS‐immunoreactive nerve fibres supports the view that NO is an important NANC‐mediator in the rabbit urethra. In contrast to several other tissues, the relaxant effect of NO in the rabbit urethra does not seem to be mediated by hyperpolarization. The network of cyclic GMP‐immunoreactive cells may constitute target cells for NO, but their function remains to be established.

Ocular inflammation induced by electroconvulsive treatment: contribution of nitric oxide and neuropeptides mobilized from C‐fibres

Electroconvulsive treatment (ECT) of rabbits produced ocular inflammation consisting of conjunctival hyperaemia, miosis and protein extravasation into the aqueous humour, reflected by the so‐called aqueous flare response (AFR); the maximal reduction in pupil size was 3.8±0.1 mm (s.e. of mean, n=16) while the maximal AFR was 28.1±2.8 (arbitrary units). ECT also caused release of substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP)‐27, ‐38 and calcitonin gene‐related peptide (CGRP). The concentrations of SP and CGRP in the aqueous humour of normal, untreated eyes were 10.6±1.4 and 117.4±12.4 pmol l−1, respectively, while the concentrations of PACAP‐27 and ‐38 were below the detection limit. After ECT the concentrations of SP, PACAP‐27, ‐38 and CGRP were 65.0±9.6, 46.9±8.4, 50.2±5.4 and 1109.9±133.1 pmol l−1, respectively (s.e. of mean, n=12). Conceivably, ECT evoked an antidromic activation of sensory neurones in the trigeminal ganglion with the consequent release of neuropeptides from C‐fibres in the uvea and the development of neurogenic inflammation. Rabbits received the nitric oxide (NO) synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 200 mg kg−1, i.v.). This pretreatment inhibited the ECT‐evoked conjunctival hyperaemia, miosis and AFR; under these circumstances the maximal reduction in pupil size was 1.9±0.1 mm while the maximal AFR was 2.7±0.9 (n=16). l‐NAME also inhibited the ECT‐evoked release of SP, PACAP‐27, ‐38 and CGRP into the aqueous humour; the concentrations of SP and CGRP were 13.2±1.5 and 204.8±33.5 pmol l−1, respectively, while PACAP‐27 and ‐38 were below the detection limit (n=12). The ECT‐evoked miosis was also inhibited by pretreatment with the tachykinin receptor antagonist d‐Pal9 spantide II (90 nmol, intravitreal injection); under these circumstances the maximal reduction in pupil size was only 0.7±0.03 mm, indicating an important role for SP in the miotic response. Pretreatment of the eye with capsaicin, which is known to cause functional ablation of C‐fibres, inhibited the conjunctival hyperaemia, miosis and AFR by 4050%; the maximal reduction in pupil size being 2.2±0.2 mm and the maximal AFR 13.8±2.1 (arbitrary units) (n=8). The results suggest (1) that ECT evokes ocular inflammation through antidromic C‐fibre activation; (2) that SP contributes to the ECT‐evoked miosis; and (3) that NO contributes to the antidromic C‐fibre activation and possibly to the vascular responses mediated by the C‐fibre transmitters.

α‐Latrotoxin‐induced transmitter release in feline oesophageal smooth muscle: focus on nitric oxide and vasoactive intestinal peptide

The effects of α‐latrotoxin (αLTX) on muscle tone, resting membrane potential, cyclic nucleotide content, and ultrastructure were examined in feline oesophageal smooth muscle, including the lower oesophageal sphincter (LOS). In circular smooth muscle strips from LOS developing active tone, αLTX (1 nm) induced a 94±3% (n=16) relaxation. Intermittent treatment with αLTX for 4 h abolished the response. Pretreatment with NG‐nitro‐l‐arginine (l‐NOARG; 0.1 mm) attenuated the relaxation. In carbachol‐contracted circular smooth muscle strips from the LOS and oesophageal body (OB), αLTX induced a 95±5% (n=6) and 73±9% (n=8) relaxation, respectively. The relaxations were attenuated by l‐NOARG, and in LOS strips, the relaxation was abolished by the combination of l‐NOARG and vasoactive intestinal peptide (VIP)‐antiserum (1:25). At resting tension in circular smooth muscle strips from the OB, αLTX induced a scopolamine sensitive contraction in the presence of l‐NOARG. In circular LOS and OB preparations, αLTX changed the resting membrane potential from −49±2 mV to −59±3 mV (n=4), and −62±2 mV to −71±3 mV (n=4), respectively. The αLTX‐induced relaxation of LOS and OB muscle was associated with a 138% and 72% increase in cyclic GMP levels, respectively. No changes in cyclic AMP levels were observed. Ultrastructural analysis of LOS and OB revealed a rich supply of nerve profiles containing small synaptic and large dense core vesicles. αLTX treatment resulted in a loss of both types of vesicle. These results suggest that αLTX induces relaxation of oesophageal circular smooth muscle associated with NO‐generation and transmitter release from synaptic vesicles. Beside NO, VIP seems to be involved in the relaxant effects of αLTX on the LOS. In addition, αLTX may have contractile effects by release of acetylcholine.

Effects of KRN2391, a novel vasodilator acting as a nitrate and a K+ channel opener, on the rabbit lower urinary tract

1. The relaxant effect of KRN2391, suggested to act both as a nitrate and a K+ channel opener, was investigated in the rabbit lower urinary tract and compared with the effects of the NO-donor SIN-1 and the K+ channel opener levcromakalim. 2. KRN2391 10(-4) M was able to relax precontracted urethral preparations by 87 +/- 4%. Corresponding values for levcromakalim 10(-4) M and SIN-1 10(-4) M were 58 +/- 8% and 103 +/- 2%, respectively. The -logEC50 values for KRN2391, SIN-1 and levcromakalim were 6.0 +/- 0.1, 4.9 +/- 0.2 and 5.8 +/- 0.2. The relaxant effect of KRN2391 on the bladder was small (29 +/- 3%). 3. The levels of cyclic GMP in the urethral preparations were significantly increased after administration of KRN2391 10(-4) M and SIN-1 10(-4) M, but not after levcromakalim 10(-4) M, the levels measured being 9.9 +/- 2.2, 20.9 +/- 5.1, and 5.2 +/- 1.0, compared to the control value, 3.7 +/- 0.5 pmol/mg protein. The levels of cyclic AMP were, however, not changed. 4. The relaxations, caused by KRN2391 in the urethral preparations, were accompanied by a hyperpolarization (14 +/- 4 mV) of the membrane potential. 5. Methylene blue 3 x 10(-5) M and glibenclamide 10(-5) M significantly reduced the relaxant effect of KRN2391 in the urethral smooth muscle. 6. We suggest that in the rabbit lower urinary tract, KRN2391 acts mainly as an NO-donor.