Viktoria Werkström

Phosphodiesterase 5 in the female pig and human urethra: morphological and functional aspects.

To characterize the distribution of phosphodiesterase 5 (PDE‐5), cGMP and cGMP‐dependent protein kinase I (PKG1), and to evaluate the effect of pharmacological inhibition of PDE‐5 in isolated preparations of pig and human urethra, as the nitric oxide (NO)/cGMP pathway generates the main inhibitory signals to reduce resistance in the bladder outlet and urethra during emptying of the bladder.

Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction

The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)‐1 and ‐2 was studied by immunohistochemistry in the pigs lower urinary tract, including bladder extramural arteries, and the oesophagogastric junction (OGJ). In isolated smooth muscle from the urethra and the OGJ, the mechanisms for CO‐induced relaxations were characterized by measurement of cyclic nucleotide levels and by responses to the guanylate cyclase inhibitor methylene blue and some K+ channel inhibitors. HO‐2 immunoreactivity was observed in coarse nerve trunks within the smooth muscle of the urethra and OGJ, and in nerve cell bodies of the enteric plexuses of the OGJ. Furthermore, the vascular endothelium of the intramural vessels of the urethra, bladder and OGJ, and the extramural vessels of the bladder, displayed HO‐2 immunoreactivity. Two different antisera against HO‐1 were used, but only one displayed immunoreactivity in neuronal structures. HO‐1 immunoreactivity, as displayed by this antiserum, was seen in nerve cells, coarse nerve trunks and varicose nerve fibres in the smooth muscle of the urethra and OGJ. Some HO‐2 and/or HO‐1 (as displayed by both HO‐1 antisera) immunoreactive cells with a non‐neuronal appearance were observed within the smooth muscle of the OGJ, bladder and urethra. In the urethral preparations, exogenously applied CO (72 μm) evoked a relaxation amounting to 76±6%. The relaxation was associated with an increase in cyclic GMP, but not cyclic AMP, content. CO‐evoked relaxations were not significantly reduced by treatment with methylene blue, or by inhibitors of voltage‐dependent (4‐aminopyridine), high (iberiotoxin, charybdotoxin) and low (apamin) conductance Ca2+‐activated, and ATP‐sensitive (glibenclamide) K+ channels. Bladder strips, and ring preparations from the extramural arteries of the bladder, did not respond to exogenously administered CO (12–72 μm). In the OGJ, exogenously applied CO evoked a relaxation of 86±6%, which was associated with an increase in cyclic GMP, but not cyclic AMP, content. Treatment with 30 μm methylene blue raised the spontaneously developed muscle tone, and reduced the maximum relaxation evoked by CO to 33±9%. Addition of 4‐aminopyridine, apamin, glibenclamide, iberiotoxin, charybdotoxin or glibenclamide had no effect on the relaxations. 4‐aminopyridine (0.1–1 mm), iberiotoxin (0.1 μm) and charybdotoxin (0.1 μm) increased the spontaneously developed tone, and a combination of charybdotoxin and apamin reduced CO‐induced (24 μm CO) relaxations. The present findings demonstrate the presence of HO in both neuronal and non‐neuronal cells in the pig OGJ and lower urinary tract. CO produces relaxation of the smooth muscle in the OGJ and urethra, associated with a small increase in cyclic GMP concentration in both regions. Relaxations evoked by CO in the urethra do not seem to involve voltage‐dependent, low and high conductance, or ATP‐dependent K+ channels. However, in the OGJ relaxations evoked by CO can be attenuated by methylene blue and a combination of charybdotoxin and apamin.

Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation

1 Non‐adrenergic, non‐cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage‐operated calcium channels (VOCCs). 2 EFS evoked frequency‐dependent and tetrodotoxin‐sensitive relaxations in the presence of propranolol (1 JIM), phentolamine (1 μM) and scopolamine (1 μIM). At low frequencies (<:12Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long‐lasting second phase. 3 Treatment with the NO‐synthesis inhibitor NG‐nitro‐L‐arginine (L‐NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (>12 Hz) only the first relaxation phase was affected. 4 Measurement of cyclic nucleotides in preparations subjected to continuous nerve‐stimulation, revealed an increase in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels from 1.3 ± 0.3 to 3.0±0.4 pmol mg−1 protein (P<0.01). In the presence of L‐NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5 Treatment with the N‐type VOCC‐inhibitor, ω‐conotoxin GVIA (0.1 μMs) reduced NO‐dependent relaxations, the effect being most pronounced at low frequencies (1‐4 Hz) of stimulation. The NO‐independent second phase of the relaxation, studied in the presence of L‐NOARG (0.3 mM) at 16–30 Hz, was however markedly reduced or abolished by ω‐conotoxin GVIA. ω‐Conotoxin MVIIC (1 μM) or ω‐agatoxin IVA (30 nM) had no effect on electrically evoked relaxations. 6 These results suggest that NANC‐nerve derived urethral relaxation in the pig consists of two apparently independent components. One is mediated by NO and associated with an increase in cyclic GMP content. The other mediator is unknown and produces relaxations not associated with changes in levels of cyclic nucleotides. The release of this mediator seems to involve the N‐type VOCC, since the relaxation was markedly reduced or abolished by ω‐conotoxin GVIA.

Morphological and functional characterization of a rat vaginal smooth muscle sphincter.

The aim of the present study was to gain information about adrenergic-, cholinergic- and non-adrenergic, non-cholinergic (NANC)- transmitter systems/mediators in the rat vagina, and to characterize its smooth muscles functionally. Tissue sections from vagina of Sprague Dawley rats were immunolabelled with antibodies against protein gene product 9.5 (PGP), synaptophysin (Syn), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Circularly cut vaginal smooth muscle preparations from the distal vagina were studied in organ baths. In the paravaginal tissue, a large number of PGP-, NOS-, TH-, VIP-immunoreactive (IR) and few CGRP-IR nerve trunks were observed, giving off branches to the smooth muscle wall. The smooth muscle wall was supplied by a large number of PGP-, Syn-, VAChT-, NPY-, NOS- and TH- IR nerve terminals, whilst only a moderate to few numbers of CGRP-, VIP- and PACAP-IR terminals were identified. Especially the distal part of the vaginal wall, where the circularly running smooth muscle was thickened into a distinct sphincter structure, was very richly innervated, predominantly by PGP- and NOS-IR terminals. Below and within the basal parts of the epithelium in the distal half of the vagina, a large number of PGP- and few NOS- and PACAP-IR varicose terminals were observed. The vaginal arteries were encircled by plexuses of nerve terminals. A large number of these were PGP-, Syn-, VAChT-, NOS-, TH-, NPY- and VIP-IR, and few were CGRP- and PACAP-IR. In isolated preparations of the distal vagina, electrical field stimulation (EFS) caused frequency-dependent contractions, which were reduced by sildenafil, tetrodotoxin (TTX) and phentolamine. In preparations contracted by norepinephrine (NA), EFS produced frequency-dependent relaxations. Pretreatment with the NOS-inhibitor NG-nitro-L-arginine, TTX, or the inhibitor of soluble guanylate cyclase, ODQ, abolished the EFS relaxations. In NE precontracted preparations, cumulative addition of sildenafil caused concentration-dependent relaxation. Carbachol contracted the strips concentration-dependently from baseline. It can be concluded that the distal part of the rat vagina forms a distinct smooth muscle sphincter, which is richly innervated by adrenergic, cholinergic and NANC nerves. The present studies suggest that in the rat the L-arginine/NO-system not only plays an important role in the regulation of vaginal smooth muscle tone, but also affects blood flow, and may have sensory functions.

Effects of diabetes on neurotransmission in rat vaginal smooth muscle

The aim of this work was to characterize the effect of experimental diabetes on neurotransmission in rat vagina. Female Sprague–Dawley rats were divided into two groups: non-diabetic controls (NDM, n=38) and diabetics (DM, n=38). DM was produced by intraperitoneal injection of streptozotocin. Eight weeks later the animals were killed, the distal part of the vagina was removed, and smooth muscle strips were prepared for functional organ bath experiments and for measurement of nitric oxide synthase (NOS) activity. In DM preparations, the EC50 value for noradrenaline (NA) was significantly increased (P<0.05) and the maximal contractile response decreased (P=0.001). In preparations precontracted with NA, the NO donor SNAP and calcitonin gene-related peptide (CGRP) caused concentration-dependent relaxations, which were significantly decreased (P<0.001) in the DM group. Electrical stimulation of nerves (EFS) caused frequency-dependent contractions, which were significantly lower in DM than in NDM strips (P<0.001). SNAP and CGRP concentration-dependently inhibited EFS evoked contractions in both NDM and DM preparations. The inhibition was significantly lower (P<0.05) in the DM group. In NDM preparations precontracted with NA, EFS evoked frequency-dependent relaxations; such relaxations were inhibited or reduced in DM. Treatment with the NOS inhibitor, L-NOARG 0.1 mM, abolished relaxations in all preparations or produced contraction in DM preparations. Calcium-dependent NOS activity was not significantly different in the DM and NDM groups. However, the DM animals showed a small but significant increase in calcium-independent NOS-activity (P<0.05). Diabetes interferes with adrenergic-, cholinergic- and NANC-neurotransmitter mechanisms in the smooth muscle of the rat vagina. The changes in the nitrergic neurotransmission are not due to reduction in NOS-activity, but seem to be due to interference with later steps in the L-arginine/NO/guanylate cyclase/cGMP system.

Inhibitory innervation of the guinea-pig urethra; roles of CO, NO and VIP

The inhibitory innervation of guinea-pig urethral smooth muscle was investigated histochemically and functionally. The distribution of immunoreactivities to haem oxygenases (HO), neuronal NO synthase (nNOS), and vasoactive intestinal polypeptide (VIP) was studied, and the functional effects of the corresponding putative transmitters, CO, NO, and VIP, were assessed. HO-2 immunoreactivity was found in all nerve cell bodies of intramural ganglia, localized between smooth muscle bundles in the detrusor, bladder base and proximal urethra. About 70% of the ganglionic cell bodies were also NOS-immunoreactive (IR), whereas a minor part was VIP-IR. Some ganglion cells exhibiting tyrosine hydroxylase (TH) activity were demonstrated. Rich numbers of NOS-IR varicose nerve terminals could be found innervating the smooth muscle of the urethra, whereas VIP-IR terminals were less numerous. A rich number of TH-IR terminals were observed. The bladder showed a similar distribution of nerves, although only a few number of TH-IR nerves could be found. In bladder preparations exposed to sodium nitroprusside, cGMP-IR cells could be seen, forming an interconnecting network with long spindle-shaped processes. The cGMP-IR cells were especially abundant in the outer smooth muscle layers of the bladder, but less numerous in the urethra. In urethral strip preparations, electrical field stimulation evoked long-lasting frequency-dependent relaxations. The relaxations were not inhibited by the NO-synthesis inhibitor, L-NOARG, or enhanced by the NO-precursor, L-arginine. The haem precursor, 5-aminolevulinic acid (5-ALA), or the inhibitor of guanylate cyclase, ODQ, did not affect the urethral relaxations. Exogenously applied NO, SIN-1, and VIP relaxed the preparations by approximately 50%, whereas the relaxation evoked by exogenous CO was minor. These results suggest that CO probably is not involved in non-adrenergic, non-cholinergic inhibitory control of the guinea-pig urethra, where a non-NO/cGMP mediated relaxation seems to be predominant.

NANC transmitters in the female pig urethra--localization and modulation of release via alpha 2-adrenoceptors and potassium channels

1 To investigate further the release, localization and identity of a non‐nitrergic mediator of smooth muscle relaxation in the female pig urethra, we studied the effects of drugs acting at α2‐adrenoceptors or K+ channels, the effects of capsaicin and chemical sympathectomy, and the actions of several transmitter candidates. 2 Electrical field stimulation (EFS; frequencies above 12 Hz) of spontaneously contracted smooth muscle strips from the female pig urethra evoked long‐lasting, frequency‐dependent relaxations in the presence of prazosin, scopolamine, and NG‐nitro‐L‐arginine. Treatment with the selective α2‐adrenoceptor agonist UK‐14 304 markedly reduced the relaxations evoked by EFS at all frequencies tested (16–30 Hz). The inhibitory effect of UK‐14 304 was completely antagonized by the α2‐adrenoceptor antagonist rauwolscine. The muscarinic M1 receptor antagonist, pirenzepine, or exogenously administered carbachol, did not have any effects on the electrically evoked relaxations. 3 Inhibition of high conductance Ca2+ activated K+ channels by iberiotoxin or charybdotoxin significantly enhanced the relaxations evoked by EFS at all frequencies. However, inhibition of voltage‐sensitive K+ channels with 4‐aminopyridine or dendrotoxin‐1, treatment with the ATP‐sensitive K+ channel blocker, glibenclamide, or treatment with the high and low conductance Ca2+ activated K+ channel blockers, tetraethylammonium chloride and apamin, had no effect on the relaxations evoked by EFS. 4 Electrically evoked relaxations were not affected by adrenergic denervation with 6‐hydroxydopamine (6‐OHDA) at any frequency. However, treatment with 6‐OHDA abolished prazosin‐sensitive electrically induced contractions, and a long‐lasting relaxation was revealed. Treatment with capsaicin, believed to damage selectively a subpopulation of primary afferent fibres, did not affect basal tone or relaxations evoked by EFS. 5 Exogenously applied vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase‐activating peptide (PACAP)‐27, PACAP‐38, adenosine, ATP and 5‐hydroxy‐tryptamine caused relaxations of the urethral preparations, whereas prostaglandin E2 and calcitonin gene‐related peptide had no effects. VIP 10‐28, α, β‐methylene‐ATP, reactive blue‐2, suramin or indomethacin did not reduce the electrically‐evoked relaxations at any frequency. However, the relaxations were slightly reduced by trypsin or α‐chymotrypsin. 6 The present results suggest that the release of the unknown mediator in the female pig urethra can be modulated via α2‐adrenoceptors and high conductance Ca2+ activated K+ channels. Furthermore, the mediator does not appear to be localized to or released from adrenergic or capsaicin‐sensitive sensory nerve‐endings. The identity of the transmitter remains to be established.

Vardenafil-induced relaxation and cyclic nucleotide levels in normal and obstructed rat urinary bladder.

To study the effects of the phosphodiesterase‐5 inhibitor, vardenafil, on contraction and cyclic nucleotide levels in isolated detrusor preparations with and without mucosa, from control rats and rats with partial urethral obstruction (PUO) and intact mucosa.

NEUROTRANSMITTER RELEASE EVOKED BY ALPHA -LATROTOXIN IN THE SMOOTH MUSCLE OF THE FEMALE PIG URETHRA

Neuronal regulation of smooth muscle tone in the female pig urethra has mainly been studied in vitro using electrical field stimulation (EFS) of nerves. Excitatory control is considered to be exerted by released noradrenaline, whereas inhibitory control is non-adrenergic non-cholinergic (NANC), and mediated by nitric oxide (NO), and an as yet unidentified agent. We investigated the functional and morphological effects of α-latrotoxin (αLTX), a spider neurotoxin believed to cause massive release of vesicle-stored neurotransmitters, on spontaneously developed urethral smooth muscle tone. The effects were compared to those of EFS and high potassium. In the presence of the NO-synthesis inhibitor Nω-nitro-L-arginine (L-NOARG: 0.3 mM) both αLTX and EFS evoked contractions. After treatment with scopolamine and phentolamine, no contraction was observed, and under these conditions αLTX and EFS induced relaxation. At low frequencies (<12 Hz), the EFS-induced relaxations were rapid, whereas at higher frequencies (>12 Hz), they were biphasic, consisting of a rapid first phase followed by a more long-lasting second phase. L-NOARG abolished the relaxations at low frequencies, as well as the first phase of the biphasic relaxation. The second phase was not affected by treatment with L-NOARG, but 0.1 µM ω-conotoxin GVIA, blocker of N-type voltage-operated calcium- channels (VOCCs), markedly reduced or abolished the response. In the presence of L-NOARG or ω-conotoxin GVIA, the αLTX-induced relaxation was significantly decreased, and the combination of L-NOARG and ω-conotoxin GVIA further reduced or abolished the relaxation. In preparationstreated with tetrodotoxin or scorpion venom, believed to inactivate nerves by acting on sodium channels, αLTX and EFS had no effects. αLTX-induced relaxation was not associated with changes in cyclic GMP or cyclic AMP content. High (80 mM) potassium solution induced a triphasic response of the preparation. A transient relaxation was followed by a restoration of tone, and then by a persistent relaxation. The persistent relaxation was slightly reduced by scorpion venom or L-NOARG, but reduced by 50% by a combination of L-NOARG and ω-conotoxin GVIA. Ultrastructural analysis of the urethral circular smooth muscle layer revealed a moderate amount of nerve profiles supplying the smooth muscle. In control preparations, the nerve profiles contained both small synaptic vesicles and large dense core vesicles. αLTX caused a major loss of both types of vesicle. The present data suggest that αLTX has the ability to release not only adrenergic and cholinergic transmitters, but also NANC mediators of relaxation, including NO, from nerve terminals in the urethra.

ATP- and adenosine-induced relaxation of the smooth muscle of the pig urethra

To investigate relaxation mechanisms for ATP and adenosine in the pig urethra, together with the possible role of ATP in nerve‐evoked urethral relaxations, as ATP is thought to cause bladder smooth muscle contraction via P2X receptors, whereas relaxation is mediated via G‐protein coupled P2Y receptors, and ATP may also induce relaxation via breakdown to adenosine.