Selim Cellek

Selective nitrergic neurodegeneration in diabetes mellitus–a nitric oxide‐dependent phenomenon

In vitro and in vivo studies have demonstrated a dysfunctional nitrergic system in diabetes mellitus, thus explaining the origin of diabetic impotence. However, the mechanism of this nitrergic defect is not understood. In the penises of streptozotocin (STZ)‐induced diabetic rats, here, we show by immunohistochemistry that nitrergic nerves undergo selective degeneration since the noradrenergic nerves which have an anti‐erectile function in the penis remained intact. Nitrergic relaxation responses in vitro and erectile responses to cavernous nerve stimulation in vivo were attenuated in these animals, whereas noradrenergic responses were enhanced. Activity and protein amount of neuronal nitric oxide synthase (nNOS) were also reduced in the penile tissue of diabetic rats. We, thus, hypothesized that NO in the nitrergic nerves may be involved in the nitrergic nerve damage, since only the nerves which contain neuronal NO synthase underwent degeneration. We administered an inhibitor of NO synthase, NG‐nitro‐L‐arginine methyl ester (L‐NAME), in the drinking water of rats for up to 12 weeks following the establishment of diabetes with STZ. Here we demonstrate that this compound protected the nitrergic nerves from morphological and functional impairment. Our results show that selective nitrergic degeneration in diabetes is NO‐dependent and suggest that inhibition of NO synthase is neuroprotective in this condition.

Y‐27632, an inhibitor of Rho‐kinase, antagonizes noradrenergic contractions in the rabbit and human penile corpus cavernosum

We have examined the effect of an inhibitor of Rho‐kinase, (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y‐27632), on the contractions elicited by noradrenergic nerve stimulation and by phenylephrine in the human and rabbit penile corpus cavernosum. In both tissues, after treatment with scopolamine (10 μM) and NG‐nitro‐L‐arginine methyl ester (L‐NAME; 300 μM), electrical field stimulation (EFS) elicited noradrenergic contractions. These contractions were inhibited by Y‐27632 in a concentration‐dependent manner. The compound caused concentration‐dependent relaxation of phenylephrine‐contracted tissues, which were treated with scopolamine (10 μM), guanethidine (10 μM) and L‐NAME (300 μM). These results suggest that Rho‐kinase is involved in noradrenergic contractile pathway in the cavernosal smooth muscle of the penis.

The Role of Intrinsic Pathway in Apoptosis Activation and Progression in Peyronie's Disease

Peyronies disease (PD) is characterized with formation of fibrous plaques which result in penile deformity, pain, and erectile dysfunction. The aim of this study was to investigate the activation of the intrinsic apoptotic pathway in plaques from PD patients. Tunica albuginea from either PD or control patients was assessed for the expression of bax, bcl-2 and caspases 9 and 3 using immunohistochemistry and by measurement of apoptotic cells using TUNEL assay. Bax overexpression was observed in metaplastic bone tissue, in fibroblasts, and in myofibroblast of plaques from PD patients. Little or no bcl-2 immunostaining was detected in samples from either patients or controls. Caspase 3 immunostaining was very strong in fibrous tissue, in metaplasic bone osteocytes, and in primary ossification center osteoblasts. Moderate caspase 9 immunostaining was seen in fibrous cells plaques and in osteocytes and osteoblasts of primary ossification centers from PD patients. Control samples were negative for caspase 9 immunostaining. In PD patients the TUNEL immunoassay showed intense immunostaining of fibroblasts and myofibroblasts, the absence of apoptotic cells in metaplasic bone tissue and on the border between fibrous and metaplastic bone tissue. Apoptosis occurs in stabilized PD plaques and is partly induced by the intrinsic pathway.

Human and rabbit cavernosal smooth muscle cells express Rho-kinase

Rho-kinase is an enzyme involved in the Ca2+-sensitizing pathway in smooth muscle cells. Inhibition of this enzyme has been recently demonstrated to elicit penile erection by relaxing cavernosal smooth muscle. We aimed to investigate the presence and activity of Rho-kinase in human cavernosal smooth muscle. Primary culture of smooth muscle cells from human and rabbit penile corpus cavernosum was developed, and cells showed characteristic myocyte morphology and α-actin immunoreactivity. The presence of Rho-kinase was demonstrated by indirect immunofluorescence and Western blotting. A specific inhibitor of Rho-kinase, Y-27632, inhibited in a concentration-dependent manner the kinase activity of the protein immunoprecipitated with anti-Rho-kinase antibody. These results demonstrate for the first time expression and activity of Rho-kinase in human penile cavernosal smooth muscle cells and suggest that these cells can provide a cellular model for the study of enzymes involved in Ca2+-sensitizing pathways.

Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral corpus cavernosum of the rabbit.

The corpus cavernosum is the erectile tissue in the penis and clitoris. Although nitrergic neurotransmission has been characterized in detail in the penile corpus cavernosum, functional studies on the inhibitory non‐adrenergic non‐cholinergic (NANC) transmission in the clitoral corpus cavernosum have been lacking. Here we demonstrate that electrical field stimulation (EFS) induces NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. These responses were inhibited by NG‐nitro‐L‐arginine methylester (L‐NAME), 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ) or tetrodotoxin. The inhibitory effect of L‐NAME was partially reversed by L‐arginine but not by D‐arginine. EFS‐induced relaxations were enhanced by an inhibitor of type V cyclic GMP phosphodiesterase, zaprinast. These results suggest that nitrergic neurotransmission is responsible for the NANC relaxation responses in the clitoral corpus cavernosum of the rabbit.

GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility.

Abstract  There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5‐hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L−1–10 μmol L−1 caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 ± 47% at 3 μmol L−1. In human‐isolated stomach, GSK962040 10 μmol L−1, erythromycin 10 μmol L−1 and [Nle13]‐motilin 100 nmol L−1, each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg−1 GSK962040 or 10 mg kg−1 erythromycin significantly increased faecal output over a 2‐h period. Together, these data show that GSK962040, a non‐motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis.

Inhibition of nitrergic relaxations by a selective inhibitor of the soluble guanylate cyclase

1 . The actions of 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ), a specific inhibitor of the soluble guanylate cyclase (SGC), were investigated in the rabbit anococcygeus muscle. 2 . ODQ (1 nM‐1 μm) inhibited in a concentration‐dependent manner the relaxations induced by electrical field stimulation (EFS; 50 V, 0.3 ms duration, 1 Hz, for 5 s, every 120 s). 3 . ODQ (1 μm) also inhibited the relaxations elicited by EFS (50 V, 0.3 ms duration, 1, 2.5, 5, 10 Hz, for 5 s) and sodium nitroprusside (SNP; 1 μm) without affecting those induced by isoprenaline (1 μm), atrial natriuretic peptide (ANP; 100 nM) or an analogue of cyclic GMP (8‐pCPT‐cyclic GMP; 500 μm). 4 . ODQ (1 μm) inhibited the elevations in the concentration of cyclic GMP induced by SNP or EFS, but not by ANP. ODQ did not affect the concentrations of cyclic AMP. 5 . Nitrergic relaxation in this tissue appears, therefore, to be mediated via activation of SGC.

5-HT4 receptor agonists enhance both cholinergic and nitrergic activities in human isolated colon circular muscle

Abstract  Previous studies have demonstrated mixed inhibitory and facilitatory effects of 5‐hydroxytryptamine‐4 (5‐HT4) receptor agonists on electrical field stimulation (EFS)‐induced responses in human isolated colon. Here we report three types of responses to EFS in human isolated colon circular muscle: monophasic cholinergic contraction during EFS, biphasic response (nitrergic relaxation during EFS followed by cholinergic contraction after termination of EFS) and triphasic response (cholinergic contraction followed by nitrergic relaxation during EFS and a tachykininergic contraction after EFS). The effects of two 5‐HT4 receptor agonists, prucalopride and tegaserod were then investigated on monophasic responses only. Each compound inhibited contractions during EFS in a concentration‐dependent manner. In the presence of Nω‐nitro‐l‐arginine methyl ester (l‐NAME) however, prucalopride and tegaserod enhanced the contractions in a concentration‐dependent manner. In strips where the tone was elevated with substance‐P and treated with scopolamine, EFS‐induced relaxations were enhanced by the two agonists. The above observed effects by the two agonists were abolished by 5‐HT4 receptor antagonist SB‐204070. The two agonists did not alter the tone raised by substance‐P in the presence of scopolamine and l‐NAME and did not affect carbachol‐induced contractions in the presence of tetrodotoxin. These results suggest that in the circular muscle of human colon, 5‐HT4 receptor agonists simultaneously facilitate the activity of neurones which release the inhibitory and excitatory neurotransmitters, nitric oxide and acetylcholine respectively.

Characterization of the non‐nitrergic NANC relaxation responses in the rabbit vaginal wall

Electrical field stimulation (EFS)‐induced non‐adrenergic non‐cholinergic (NANC) relaxation responses in the rabbit vaginal wall were investigated. These NANC responses were partially inhibited with the nitric oxide synthase (NOS) inhibitors NG‐nitro‐L‐arginine methyl ester (L‐NAME; 500 μM), NG‐nitro‐L‐arginine (300 μM) or N‐iminoethyl‐L‐ornithine (500 μM) or the selective soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ, 10 μM). Application of L‐NAME and ODQ concomitantly did not increase the degree of inhibition. L‐NAME or ODQ were observed to be more effective at low frequencies. The resistant part of the responses was more pronounced at higher frequencies and was completely inhibited by tetrodotoxin (1 μM). Exogenous application of the peptides vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP‐27 and PACAP‐38), peptide histidine methionine (PHM), peptide histidine valine (PHV), helospectin‐I or ‐II induced a relaxation response. Calcitonin gene‐related peptide or substance P did not cause any relaxation. The peptidase α‐chymotrypsin (type II; 2 units ml−1) did not affect non‐nitrergic NANC responses, although it did inhibit relaxation responses elicited by exogenous VIP, PACAP‐27, PACAP‐38, PHM, PHV, helospectin‐I or ‐II. K+ channel inhibitors apamin (1 μM) or charybdotoxin (100 nM) when used alone or in conjunction did not affect non‐nitrergic NANC responses. The non‐nitrergic NANC responses were not associated with any increase in intracellular cyclic adenosine‐3′, 5′‐monophosphate (cyclic AMP) or cyclic guanosine‐3′, 5′‐monophosphate (cyclic GMP) concentrations. The peptide‐induced relaxations were all associated with increases in cyclic AMP concentrations. These results suggest that a neuronal factor elicits non‐nitrergic NANC responses in the rabbit vaginal wall. The identity of this factor remains to be established.

Investigation of cavernosal smooth muscle dysfunction in low flow priapism using an in vitro model

The effects of hypoxia (pO2: 50 mmHg), acidosis (pH: 6.9) or glucopenia (absence of glucose) in vitro on the tone of the rabbit corpus cavernosum were investigated. The recovery of smooth muscle contractility following exposure to these conditions was also assessed. Hypoxia, acidosis or glucopenia alone or in combination showed a sustained reduction in the tone. Reperfusion of tissue strips showed complete recovery of smooth muscle tone for all conditions except when hypoxia and glucopenia were combined or when hypoxia, glucopenia and acidosis were used in combination. Incomplete recovery of tone was associated with a significant reduction in tissue ATP concentrations and an increase in the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive nuclei. This indicates that following reversal of hypoxia, acidosis and glucopenia, failure of conventional α-adrenergic agonists to produce tumescence in low flow priapism is associated with irreversible smooth muscle cell dysfunction, which is linked to ATP reduction and smooth muscle cell death.