Neide H. Jurkiewicz

Evidence for participation of nitric oxide in excitatory neurotransmission in rat vas deferens

A depression of the fast, non-adrenergic, and also of the slow, adrenergic, components of muscle contraction in response to intramural nerve stimulation was induced by the blocker of nitric oxide synthase NG-nitro-L-arginine methyl ester (L-NAME), in rat vas deferens. Effects of exogenous noradrenaline or ATP were not reduced by L-NAME. However, L-arginine also caused an inhibition of electrically induced effects in most of the preparations, contrary to the expectations for a precursor of nitric oxide synthesis. In spite of these difficulties L-arginine antagonized the action of L-NAME. These results indicate that nitric oxide is involved in excitatory nerve-muscle transmission in vas deferens.

Functional properties of agmatine in rat vas deferens

Experiments were performed with rat vas deferens to verify whether agmatine, an endogenous ligand for adrenoceptors and imidazoline receptors, can influence sympathetic neurotransmission, with respect to contractions induced by transmural nerve stimulation, contractions induced by exogenous noradrenaline, and overflow of endogenous noradrenaline. It was shown that agmatine (a) caused a dose-dependent potentiation of electrically induced twitches, up to about 70% in relation to controls, (b) shifted to the right the inhibitory concentration-response curves for clonidine on electrically induced twitches, indicating competitive antagonism at presynaptic alpha-adrenoceptors, with a pA2 value of 4.12 +/- 0.10, (c) shifted to the right the concentration-response curves for noradrenaline-induced contractions, indicating competitive antagonism at postsynaptic alpha-adrenoceptors as well, with a pA2 value of 4.03 +/- 0.10, and (d) caused a dose-dependent increase of KCI-induced overflow of noradrenaline, up to about 90% in relation to controls. It is concluded that agmatine has multiple effects on sympathetic neurotransmission in rat vas deferens.

In vitro denervation of the rat vas deferens through hypothermic storage

1 The rat vas deferens was excised, stored at 4–6°C and tested after 24, 48, 72 or 96 h for its contractile activity and for the presence of innervation. 2 The maximal contractile capacity of the vas, tested through cumulative concentrations of barium chloride (3 × 10−2 m) was progressively reduced from about 110 mm to about 63 mm after 72 h, without further decay after 96 h. Spontaneous rhythmic contractions were practically absent. 3 A loss of endogenous pools of catecholamines was indicated by four parameters: (a) a decline of about 80% after 24 h and of more than 95% after 48 h of the contractile effect of the indirect sympathomimetic agonist tyramine; (b) a fall of about 20%, 50% and 85% on the concentration of noradrenaline, respectively after 24, 48 and 72 h; (c) a fall of about 25% and 90% after respectively 24 and 48 h, of the activity of dopamine‐β‐hydroxylase (DBH); (d) a decline of noradrenaline‐induced histofluorescence on cross sections of the vas. 4 A loss of neuronal uptake capacity was indicated by: (a) a progressive variation of the apparent affinity for adrenaline, expressed as pD2 values, that increased by about 1.5 log units (corresponding to a 30 fold potentiation) after 72 h, and (b) a reduction of the ability of cocaine to potentiate the contractile effects of adrenaline. 5 The pD2 values for barium chloride, 5‐hydroxytryptamine (5‐HT) and histamine were not significantly changed, while the corresponding value for acetylcholine was slightly but significantly reduced by about 0.8 log units. 6 The maximal heights of concentration‐response curves for noradrenaline, acetylcholine, histamine and 5‐HT were reduced by 42–66% in relation to controls. However, when this reduction was measured in relation to the corresponding barium effect, by means of the relative responsiveness ratio (p), a small though significant increase was observed for noradrenaline, and a fall for the other drugs. 7 It is concluded that: (1) the values for the various biochemical and pharmacological parameters decline at different rates, though revealing altogether that denervation is completed by at least 85% after 72 h of hypothermic storage; (2) two of the results, i.e., the lack of spontaneous rhythmic contractions and the lack of increased contractile effects for acetylcholine, 5‐HT and histamine, indicate that in these conditions the vas is devoid of the so‐called nonspecific signs of denervation.

Low dihydropyridine receptor density in vasa deferentia of castrated rats.

Radioligand binding studies in crude membrane preparations of vasa deferentia of normal rats, with the 1,4‐dihydropyridine (+)‐[3H]‐PN200–110 (isradipine) showed typical saturation isotherms. The binding exhibited a KD of 259 ± 60 pm and Bmax of 144 ± 20 fmol mg−1 protein. The low KD and the stereoselective displacement of (+)‐[3H]‐PN200–110 binding by (+)‐ and (−)‐PN200–110 and by nifedipine suggests that these tissues contain dihydropyridine receptors probably coupled to voltage‐sensitive, L‐type calcium channels. In membrane preparations from vasa deferentia from rats castrated 30 days previously the maximum specific binding was 25 ± 10 fmol mg−1 protein, representing only 11% of total binding; thus, the calculation of reliable KD values was not feasible. These findings suggest that a testicular hormone, possibly testosterone, plays an important role in the regulation of dihydropyridine‐sensitive, voltage‐dependent calcium channels in the rat vas deferens.

Decreased density of binding sites for the Ca2+ channel antagonist [3H]isradipine after denervation of rat vas deferens.

Radioligand binding assays were performed with the selective antagonist of dihydropyridine-sensitive Ca2+ channels [3H]PN200-110 (isradipine) in rat vas deferens, before and 7 days after denervation, and data were compared with those obtained for K(+)-induced contractions, which are Ca(2+)-dependent. The density (Bmax) of dihydropyridine binding sites was decreased to almost one-third of its normal value after denervation. The respective affinity (KD) was not significantly changed. In addition, it was observed that the K(+)-induced tonic contraction, which corresponded to 55 +/- 2% of the respective phasic contraction, was decreased to 41 +/- 3% after denervation. It is assumed that the decreased density of Ca2+ channels causes a decrease in K(+)-induced influx of Ca2+ and consequently of the corresponding tonic contraction. These results indicate that autonomic innervation can regulate the density of dihydropyridine-sensitive Ca2+ channels in the rat vas deferens.

TTX-sensitive Na+ and nifedipine-sensitive Ca2+ channels in rat vas deferens smooth muscle cells

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.

Nantenine blocks muscle contraction and Ca2+ transient induced by noradrenaline and K+ in rat vas deferens

The effect of nantenine, an aporphine alkaloid isolated from Ocotea macrophylla H.B.K., was studied on contractions and Ca(2+) translocation induced by noradrenaline, Ca(2+), or K(+) in the isolated rat vas deferens from reserpinized animals. Concentration-response curves of calcium chloride (CaCl(2)) were performed in the vas deferens, in a Ca(2+)-free nutrient solution, using potassium chloride (KCl, 80 mM) as a depolarizing agent. In these conditions, nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly reduced the maximum contractions (E(max)) of Ca(2+) (IC(50)=2.6 x 10(-4) M) and noradrenaline (IC(50)=2.9 x 10(-4) M). The contractile responses were totally recovered after the withdrawal of nantenine. In addition, experiments performed to measure simultaneously the contraction and the increase of intracellular Ca(2+) induced by noradrenaline (10(-5) M) or KCl (80 mM) showed that nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly decreased both effects. The results suggest that a reversible block of Ca(2+) entry could be involved on the non-competitive-like antagonism of nantenine in rat vas deferens.

Functional Change of the Balance between α1A and α1B Adrenoceptor Populations after Transplantation of the Vas Deferens to the Intestine

The a,-adrenoceptor involved in contractions to norepinephrine (NE) in the rat vas deferens has been identified as alA by means of both transductional and operational methods.’,2 Our objective was to verify, by means of the determination of apparent affinity constants of antagonists (pA2 values) and of the effects of chloroethylclonidine (CEC) and of the calcium antagonist isradipine, if this receptor population is changed after transplantation of the vas to the intestinal wall for seven days.-’

Contractile responses of the rat vas deferens after epithelium removal

The purpose of the present investigation was to verify the role of the epithelium in the functional response of the rat vas deferens. Our results showed that the contractile effect of cumulative doses of clonidine (3.10(-5)-3.10(-3)) was increased after the removal of the epithelium. The effect of clonidine in epithelium-free vas deferens returned to normal values when an isolated epithelium from another vas deferens was added to the organ bath, showing that the epithelium is responsible for this increase of maximum effect for clonidine. Drugs functionally or structurally related to clonidine, such as oxymetazoline, alpha-methylnorepinephrine and moxonidine, did not have their dose-response curves altered. The curves for other contractile agents, such as noradrenaline, acetylcholine, ATP, 5HT, bradykinin and histamine, or the relaxation induced by isoprenaline and forskolin were also not modified. Electrically-induced contractions at frequencies from 0.1 to 20 Hz and the mechanism of negative feed-back, brought about by clonidine (10(-10)-10(-8) M) through pre-synaptic alpha2-adrenoceptors, were not changed after the removal of epithelium. In conclusion, a significant function of the epithelium in the contractility of the rat vas deferens was demonstrated for clonidine, but not for other agonists.

Increase of angiotensin-converting enzyme activity and peripheral sympathetic dysfunction could contribute to hypertension development in streptozotocin-induced diabetic rats

Diabetes augments the risk of hypertension. Although several factors have been implicated in the development of such hypertensive state, we designed this study to investigate blood pressure development, the activity of angiotensin-converting enzyme (ACE) in blood as well as sympathetic neurotransmission in the vas deferens of diabetic rats. We used streptozotocin (STZ)-induced diabetic rats (60 mg/kg) in order to evaluate the systolic blood pressure (SBP), ACE activity and peripheral sympathetic neurotransmission. We observed the following changes of parameters: increase of SBP, decrease of heart rate, augmentation of plasma ACE activity, enhancement of phasic and tonic vas deferens contractions elicited by electrical stimulation at 5 Hz, increase of maximal response to noradrenaline (NA) and decrease of adenosine triphosphate (ATP)-elicited contraction of vasa deferentia. The results reveal that in the development of hypertension in diabetic rats, augmentation of circulating ACE activity precedes the sympathetic dysfunction. Additionally, it seems that the purinergic and noradrenergic neurotransmission is compromised.