Lars Ny

Defective smooth muscle regulation in cGMP kinase I‐deficient mice

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP‐dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross‐activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP‐dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI‐deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function

The vasodilator‐stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP‐ and cGMP‐dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP‐deficient mice had normal agonist‐induced contraction, and normal cAMP‐ and cGMP‐dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP‐ and cGMP‐mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP‐ and cGMP‐dependent effects in platelets, such as inhibition of agonist‐induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide‐dependent mechanisms are operating during platelet activation: a VASP‐independent mechanism for inhibition of calcium mobilization and granule release and a VASP‐dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

Pitfalls using metalloporphyrins in carbon monoxide research

The proposal that endogenously produced carbon monoxide (CO) may act as a biological messenger has remained controversial. Carbon monoxide is generated by haem oxygenase isoenzymes in the degradation of haem-containing molecules. Certain metalloporphyrins, which are inhibitors of haem oxygenase, have been widely used as pharmacological tools in order to establish a messenger role for CO in the brain and periphery. However, increasing evidence shows that many metalloporphyrins are also associated with a large range of undesired effects, which make the interpretation of results using such compounds very uncertain. In this article, Lars Grundemar and Lars Ny evaluate the properties and describe the nonselective effect profile of such metalloporphyrins.

CHOLINERGIC NERVES IN HUMAN CORPUS CAVERNOSUM AND SPONGIOSUM CONTAIN NITRIC OXIDE SYNTHASE AND HEME OXYGENASE

PURPOSE To characterize the distribution of cholinergic nerves in the human corpus cavernosum (CC) and spongiosum (CS) using antibodies to the vesicular acetylcholine transporter (VAChT), and to compare this distribution to those of other transmitters/mediators or transmitter/mediator generating enzymes (heme oxygenases: HO-1 and HO-2; neuronal and endothelial NO synthases: nNOS and eNOS; vasoactive intestinal polypeptide: VIP; and tyrosine hydroxylase: TH), and to investigate NO- and carbon monoxide (CO)-mediated effects. MATERIALS AND METHODS Immunocytochemistry, confocal laser scanning microscopy, radioimmunoassay, and functional in vitro studies. RESULTS Along strands of smooth muscle in the CC and CS, rich numbers of VAChT-, nNOS-, VIP-, TH-, and very few HO-1-immunoreactive (-IR) nerve fibers were observed. Immunoreactivities for VAChT and nNOS, VAChT and VIP, and nNOS and VIP, were generally found in the same varicose nerve terminals. TH-IR nerve fibers or terminals did not contain immunoreactivities for VAChT, NOS or VIP. In the endothelium lining penile arteries, immunoreactivities for eNOS, HO-1, and HO-2 were detected. Single endothelial cells, lining the sinusoidal walls of the CC and CS, were found also to contain eNOS and HO-immunoreactivities. Noradrenaline (NA)-contracted preparations of CC and CS were relaxed by NO, CO, carbachol and by electrical stimulation of nerves. Inhibition of NO synthesis abolished electrically- and carbachol-induced relaxation. In NA-activated strips, relaxation induced by exogenously applied NO, but not those by CO, were accompanied by increases in intracellular levels of cyclic GMP. CONCLUSIONS VAChT, NOS and VIP are found in the same nerve terminals within the human CC and CS, suggesting that these terminals comprise a distinct population of parasympathetic, cholinergic nerves. Endothelially derived NO and the HO/CO system may have a complementary role in penile erection.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP‐dependent protein kinase I

Guanosine 3′, 5′‐cyclic monophosphate (cyclic GMP)‐dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP‐ and vasoactive intestinal polypeptide (VIP)/adenosine 3′, 5′‐cyclic monophosphate (cyclic AMP)‐signalling pathways in the gastric fundus of wild type and cGKI‐deficient mice. Using immunohistochemistry, similar staining patterns for NO‐synthase, cyclic GMP‐ and VIP‐immunoreactivities were found in wild type and cGKI‐deficient mice. In isolated, endothelin‐1 (3 nM–3 μM)‐contracted, muscle strips from wild type mice, electrical field stimulation (1–16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI‐deficient mice only the slowly developing relaxation was observed. The responses to the NO donor, SIN‐1 (10 nM–100 μM), and to 8‐Br‐cyclic GMP (10 nM–100 μM) were markedly impaired in strips from cGKI‐deficient mice, whereas the responses to VIP (0.1 nM–1 μM) and forskolin (0.1 nM–1 μM) were similar to those in wild type mice. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP‐pathway can exert their effects independently of cGKI.

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.

Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter

1 In the feline lower oesophageal sphincter (LOS), the distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)−‐1 and −2 was studied by immunohistochemistry and confocal microscopy, the HO activity was measured and the possible role for CO as a mediator of relaxation was investigated. 2 HO‐2 immunoreactivity was abundant in nerve cell bodies of the submucosal and myenteric plexus. Approximately 50% of the HO‐2‐containing myenteric cell bodies were also nitric oxide synthase‐ and vasoactive intestinal peptide (VIP)‐immunoreactive. In addition, HO‐2 immunoreactivity was seen in nerve fibres, in non‐neuronal cells dispersed in the smooth muscle and in arterial endothelium. HO‐1 immunoreactivity was confined to non‐neuronal cells in the smooth muscle, similar to those positive for HO‐2. 3 Activity of HO, measured as CO production, was observed in LOS homogenates at a rate of 1.00±0.05 nmol mg−1 protein h−1. This production was inhibited by the HO inhibitor, zinc protoporphyrin‐IX (ZnPP). 4 In isolated circular smooth muscle strips of LOS, developing spontaneous tone, exogenously administered CO evoked a concentration‐dependent relaxation reaching a maximum of 93±3%. This relaxation was accompanied by an increase in cyclic GMP, but not cyclic AMP levels. The relaxant response was attenuated by methylene blue, but unaffected by tetrodotoxin. Repeated exposure to CO resulted in a progressive reduction of the relaxant response. 5 ZnPP caused a rightward‐shift of the concentration‐response curves for the relaxant responses to VIP, peptide histidine isoleucine, and pituitary adenylate cyclase activating peptide 27. 6 ZnPP and tin protoporphyrin‐IX (another inhibitor of HO) did not affect nonadrenergic, noncholinergic relaxations induced by electrical field stimulation. Nor did ZnPP affect relaxations induced by 3‐morpholino‐sydnonimine or forskolin. 7 The present findings, showing localization of HO immunoreactivity to both neuronal and non‐neuronal cells of the feline LOS, ability of LOS to produce CO and a relaxant effect of CO in circular LOS muscle, suggest a role for CO as a peripheral messenger.

Morphological relations between haem oxygenases, NO-synthase and VIP in the canine and feline gastrointestinal tracts

Carbon monoxide (CO), produced by haem oxygenase (HO), has been suggested as a messenger molecule in the central and peripheral nervous systems. In the present study, we have investigated the occurrence of the two isoforms of HO, HO-2 and HO-1 in the canine and feline gastrointestinal tracts, including the small and large intestine and the gastrointestinal sphincters. An abundance of nerve cell bodies that contained immunoreactivity for HO-2 was found in the submucosal and myenteric plexuses. HO-2 immunoreactivity was frequently co-localized with nitric oxide synthase (NOS) or vasoactive intestinal peptide (VIP) immunoreactivities and was also observed in some nerve fibres, certain non-neuronal cells dispersed among smooth muscle bundles, and in vascular endothelium. The antiserum against HO-1 revealed immunoreactivity in nerve cell bodies in the enteric plexuses, in nerve fibres and in non neuronal cells in the smooth muscle layers. Some of the nerve structures were also NOS- or VIP-immunoreactive. These results demonstrate the presence of HO isoenzymes in nerves and other structures of the canine and feline gastrointestinal tracts and support the view that CO may have a role as a messenger molecule in the enteric nervous system.

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.